for Supercritical Fluid Chromatography (SFC) · 2007-07-06 · 3 Title: Correlation of...

Literature citations for Supercritical Fluid Chromatography (SFC)

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Title: Determination of alcohol polyether average molar oligomer value/distribution via supercritical fluid chromatography coupled with UV and MS detection

Authors: Hoffman BJ., Taylor LT., Rumbelow S., Goff L., Pinkston JD.

Journal: J. Chromatogr. A 2004 1043 285–290

Address: Virginia Tech Department of Chemistry Blacksburg, VA 24061, USA

Abstract: Supercritical fluid chromatography (SFC) was used for the analysis of ethoxylated and propoxylated surfactants. Samples were derivatized to phenylated silyl ethers with a disilazane–chlorosilane mixture. Addition of a phenyl group to the surfactant allowed UV-absorbance detection of each oligomer. Acetonitrile and methanol were evaluated as mobile phase modifiers. Better peak shape was realized with methanol-modified CO2 on an octadecyl silica bonded phase than with acetonitrile-modified CO2. Peak assignments were made via SFC coupled with electrospray ionization–mass spectrometry (ESI–MS) in the positive ion mode. A sulfonamide-embedded alkyl stationary phase was also evaluated for separation of the derivatized samples. SFC–UV and SFC–ESI–MS data were jointly used for calculation of average molar oligomer values which were then compared to values calculated from 1H NMR data of non-derivatized samples. The derivatization or separation method using the sulfonamide embedded phase required no preliminary cleanup and yielded reproducible oligomer values that were consistent with those of the manufacturer’s nominal values.

Title: Analysis of the sulfomycin

component of alexomycin in animal feed by enhanced solvent extraction and supercritical fluid chromatography

Authors: Ashraf-Khorassani M., Taylor LT., Marr JGD.

Journal: J. Biochem. Biophys. Methods 2000 43 147–156

Address: Virginia Tech Department of Chemistry Blacksburg, VA 24061, USA

Abstract: Enhanced solvent extraction (ESE) was used to isolate components of alexomycin from different types of animal feed samples. It was demonstrated that ESE was more economical as regards money and, for this particular matrix, twice as fast as supercritical fluid extraction (SFE) even though it was demonstrated that alexomycin can be extracted from feed quantitatively using both ESE and SFE. Supercritical fluid chromatography was used to separate alexomycin from other co-extractives of feed since liquid chromatography was unable to isolate the alexomycin for quantification purposes. Our results also showed that the concentration of alexomycin in an extruded sample was approximately half of the concentration which was originally added to the feed.

Literature Citations for Supercritical Fluid Chromatography

Title: Separation of ketoconazole

enantiomers by chiral subcritical-fluid chromatography

Authors: Bernala JL., del Nozala MJ., Toribioa L., Montequib MI., Nieto EM.

Journal: J. Biochem. Biophys. Methods 2000 43 241–250

Address: University of Valladolid Department of Analytical Chemistry, Faculty of Sciences Prado de la Magdalena s/n E-47005 Valladolid, Spain

Abstract: The separation of ketoconazole enantiomers by subcritical-fluid chromatography using an amylose-based column is described. Drastic changes in the resolution have been obtained for the different organic modifiers evaluated, with ethanol providing the best results. Other chromatographic parameters such as temperature, pressure and flow-rate have also been studied. The best results in terms of resolution and analysis time were obtained using 30% ethanol (containing 0.1% triethylamine and 0.1% trifluoroacetic acid), a pressure of 300 bar, a temperature of 358oC and a flow-rate of 3 ml /min. Under these conditions the ketoconazole enantiomers are resolved in a short time (less than 7 min) and with high resolution (4.29).

Title: Development of an amperometric

detector for packed capillary column supercritical fluid chromatography

Authors: Wallenborg SR., Markides KE., Nyholm L.

Journal: Anal. Chem. 1997 69 439-445

Address: Uppsala University Department of Analytical Chemistry P.O. Box 531 S-751 21 Uppsala, Sweden

Abstract: Amperometric detection at a naked platinum microelectrode is shown to be compatible with pressure-programmed packed capillary column supercritical fluid chromatography (SFC) using carbon dioxide modified with as little as 1% acetonitrile. Amperometric detection in modified carbon dioxide is, hence, possible without addition of salts, which otherwise may limit the stability of the chromatographic system. The detection, which can been based on either oxidations or reductions, is also compatible with the use of methanol as a sole modifier. Picogram amounts of ferrocene could be detected after separations both at a constant pressure and under pressure-programmed conditions. The detection limit for ferrocene, at a constant pressure of 200 atm, was found to be approximately 20 pg. The response of the amperometric detector was observed to increase with decreased mobile phase density and increased amount of added modifier. The injection of increasing amounts of analytes resulted in increased peak tailing, most likely due to the limited solubility of the oxidation products in the mobile phase. The influence of this effect was, however, small for the amounts of analytes relevent in capillary column SFC.

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Title: Correlation of supercritical fluid extraction with supercritical fluid chromatography in aqueous Matrixes

Authors: Yu T., Luo SK., Chen SJ.


Address: National Chiao Tung University Department of Applied Chemistry Hsinchu, Taiwan, Republic of China

Abstract: A mathematical model is presented for correlating supercritical fluid chromatography (SFC) in aqueous stationary phases with supercritical fluid extraction (SFE) in aqueous matrixes. A solar coaxial countercurrent chromatography apparatus was used for the SFC and SFE experiments. The SFE extraction vessel, i.e., the column for SFC, was mathematically divided into limited layers. During extraction, each layer was considered to undergo a chromatographic process. The plate heights of all the layers were regarded equal throughout the column because pressure drops in the system were negligible. Each layer’s chromatographic capacity factor and peak width were calculated using true SFC experimental data, and the sum of all these peak distributions as a function of time gave the extraction efficiency. Accordingly, the SFE analyte recovery curve could be simulated using SFC data and this model. Since SFC operations are more straightforward than SFE operations, SFE optimization may be more easily achieved using this mathematical correlation. The simulated analyte data of large capacity factors matched the experimental results very well. Deviations gradually became greater as analyte capacity factors were decreased. A rationale is proposed that satisfactorily interprets this deviation trend.

Title: Direct analysis of gramicidin

double helices using packed column supercritical fluid chromatography

Authors: Zhang J., Thurbide KB.


Address: University of Calgary Department of Chemistry 2500 University Drive, N.W., Calgary, Alta., Canada T2N 1N4

Abstract: Direct analysis of the monomeric and four double helical dimeric conformations of gramicidin has been achieved using packed column supercritical fluid chromatography (pSFC). Using a PRP-1 polymeric column and typical conditions of 40◦C column temperature, 25MPa column pressure, and 35% n-pentanol modifier addition, all of the gramicidin conformers were readily separated. To evaluate the method, the dynamic characteristics of the monomer and dimer species were monitored as a function of solvent type, incubation time, solvent temperature, and initial concentration. The findings agree with those previously obtained by other methods but also yield new information about the relative amounts of two closely related dimers (species 1 and 2) as well as the simultaneous changes in the full dimer/monomer distribution. Results indicate that the developed pSFC method can be an informative complimentary tool for readily monitoring changes in the full profile of gramicidin species present in different environments.

Title: Chiral supercritical fluid Abstract:

chromatography/tandem mass spectrometry for the simultaneous determination of pindolol and propranolol in metabolic stability samples

Authors: Hsieh Y, Favreau L, Cheng KC., Chen J.

Journal: Rapid Commun. Mass Spectrom. 200519 3037–3041

Address: Schering-Plough Research Institute Drug Metabolism and Pharmacokinetics Department Kenilworth, NJ 07033, USA.

A chiral packed-column supercritical fluid chromatographic (pSFC) system coupled to tandem mass spectrometer (MS/MS) for the rapid measurements of (R,S)-propranolol and ( )-pindolol in metabolic stability samples was developed. The effects of the eluent flow rates and compositions, as well as of the nebulizer temperature, on the chromatographic performance and the ionization efficiency of the analytes in positive ion mode under pSFC conditions were studied. The ionization mechanism of the analytes in the CO2/methanol atmospheric pressure chemical ionization (APCI) environments with or without the use of an additive was studied. The chiral pSFC-APCIMS/MS approach requiring approximately 2 min per sample was applied for the simultaneous determination of two pairs of racemic drugs in in vitro samples at low nanogram per milliliter concentrations.

Title: Increasing bioanalytical

throughput ysing pcSFC-MS/MS: 10 minutes per 96-well plate

Authors: Hoke SH., Tomlinson JA., Bolden RD, Morand KL, Pinkston JD., Wehmeyer KR.


Address: The Procter & Gamble Company Health Care Research Center P.O. Box 8006 Mason, Ohio 45040, USA.

Abstract: The utility of packed-column supercritical, subcritical, and enhanced fluidity liquid chromatographies (pcSFC) for high-throughput applications has increased during the past few years. In contrast to traditional reversed-phase liquid chromatography, the addition of a volatile component to the mobile phase, such as CO2, produces a lower mobile-phase viscosity. This allows the use of higher flow rates which can translate into faster analysis times. In addition, the resulting mobile phase is considerably more volatile than the aqueous-based mobile phases that are typically used with LC-MS, allowing the entire effluent to be directed into the MS interface. High-throughput bioanalytical quantitation using pcSFC-MS/MS for pharmacokinetics applications is demonstrated in this report using dextromethorphan as a model compound. Plasma samples were prepared by automated liquid/liquid extraction in the 96-well format prior to pcSFC-MS/MS analysis. Three days of validation data are provided along with study sample data from a patient dosed with commercially available Vicks 44. Using pcSFC and MS/MS, dextromethorphan was quantified in 96-well plates at a rate of 10 min/plate with average intraday accuracy of 9% or better. Daily relative standard deviations (RSDs) were less than 10% for the 2.21 and 14.8 ng/mL quality control (QC) samples, while the

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RSDs were less than 15% at the 0.554 ng/mL QC level.

Title: Coupled achiral/chiral column techniques in subcritical fluid chromatography for the separation of chiral and nonchiral compounds

Authors: Phinney KW., Sander LC, Wise SA.


Address: National Institute of Standards and Technology Chemical Science and Technology Laboratory Analytical Chemistry Division, Gaithersburg, Maryland 20899-0001, USA.

Abstract: A multicolumn approach was developed to address the limited achiral selectivity of chiral stationary phases. Groups of structurally related compounds, including â-blockers and 1,4-benzodiazepines, were separated using coupled achiral/chiral stationary phases under subcritical fluid conditions. The achiral selectivity of amino and cyano stationary phases was used to modify the resolution of compounds on a Chiralcel OD chiral stationary phase by combining the achiral and chiral columns in series. In the case of the benzodiazepines, separation of achiral compounds was performed concurrently with the enantioseparation of chiral molecules. The separation of components of a multidrug cough and cold medication was also demonstrated on a cyano column coupled with a Chiralpak AD chiral stationary phase. The use of modified carbon dioxide eluents eliminated the mobile phase incompatibility problems associated with column coupling in liquid chromatography and incorporated the high efficiency of sub- and supercritical fluid chromatography.

Title: Factors That Control Successful

Entropically Driven Chiral Separations in SFC and HPLC

Authors: Stringham RW., Blackwell JA.


Address: The DuPont Merck Pharmaceutical Company CPR&D Chambers Works PRF (S1) Deepwater, New Jersey 08023-0999, USA

Abstract: With temperature increases, selectivity of chiral separations decreases until enantiomers coelute at an isoelution temperature. Above this temperature, elution order should reverse and selectivity will increase with temperature. In this region, separation is termed “entropically driven”. Entropically driven chiral separations hold the promise of being able to concurrently increase selectivity and column efficiency by means of increased temperature. The ability to achieve such separations is hindered by high isoelution temperatures. The isoelution temperature is determined by a balance of enthalpic and entropic contributions. A variety of mobile phase modifiers are evaluated for their ability to moderate these contributions. Results suggest that more use should be made of nonalcohol modifiers. The major barrier to entropically driven separations was found to be the nonspecific retention increase that is characteristic when the critical temperature is traversed. Use of hexane in place of CO2 shifts the position of the retention increase away from the temperature range

used in this study, and dramatically successful entropically driven chiral separations are obtained.

Title: Separation of substituted aromatic isomers with porous graphitic carbon in subcritical fluid chromatography

Authors: West C., Lesellier E.


Address: LETIAM IUT d'Orsay EA 3343, Groupe de Chimie Analytique de Paris Sud, Plateau du Moulon, F-91400 Orsay, France.

Abstract: The ability of porous graphitic carbon (PGC) to separate structural isomers has been reported in high-performance liquid chromatography (HPLC). This paper presents studies carried out in subcritical fluid chromatography (SubFC). Various polar and nonpolar modifiers were added to the carbon dioxide mobile phase, in proportions ranging from 5 to 40%. The effects of both the nature and the percentage of the modifier on aromatic isomer separations were studied. Two types of selectivity behaviour appear. The first one, related to steric recognition, is due to the number of contact points between the compounds and the flat surface of PGC. In this case, retention orders are often identical to that reported in HPLC. The second is related to the favourable interaction between the polar moieties of the solutes and the stationary phase. In this case, the retention and selectivity strongly depend on the mobile phase composition. Thus, the separations obtained are greatly enhanced, compared to those obtained in HPLC. The retention and selectivity variations observed when the composition of the mobile phase is changed are discussed based on linear solvation energy relationships (LSERs). Practical applications are presented, namely benzene, toluene, ethylbenzene and xylenes (BTEX) and flavour molecules separations.

Title: Capillary supercritical fluid

chromatography/mass spectrometry of phenolic mannich bases with dimethyl ether modified ethane as the mobile phase

Authors: Fuchslueger U., Socher G., Grether HJ., Grasserbauer M.


Address: Ciba Specialty Chemicals Inc.

Abstract: The analysis of phenolic Mannich bases which are used as hardeners and accelerators for epoxy resinssby capillary supercritical fluid chromatography (SFC) with dimethyl ether modified ethane as the mobile phase is described. The elution properties of several different mobile phases with respect to amines are shown. SFC with UV detection is coupled via a custom-built interface to a mass spectrometer with atmospheric pressure chemical ionization. Two technically important Mannich bases prepared by different production processes are characterized and compared with respect to their byproducts. The role of dimethyl ether during the ionization process and the fragmentation of phenolic Mannich bases is discussed.

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Performance Polymers K-402.5.03 CH-4002 Basel, Switzerland

Title: Packed-column supercritical fluid

chromatography for the analysis of isosorbide-5-mononitrate and related compounds in bulk substance and tablets

Authors: Gyllenhaal O., Karlsson A.

Journal: J. Biochem. Biophys. Methods 2000 43 135-146

Address: AstraZeneca R&D Molndal S-431 83 Molndal, Sweden

Abstract: We describe a packed-column supercritical fluid chromatographic method that can be used for the analysis of isosorbide-5-mononitrate (5-ISMN) bulk substance and the 5-ISMN content of Imdur tablets. The method is based on methanol-modified carbon dioxide as the mobile phase and porous graphitized carbon (PGC, Hypercarb) as column support at 408C and 100 bar back pressure. The method makes it possible to simultaneously determine 5-ISMN and related 2 compounds. In order to elute NO with acceptable retention time a quarternary ammonium 3 hydrogen sulfate salt is added to the methanol modifier. An almost linear increase of the retention time with increasing carbon content of the counter ion was found. Tetramethyl ammonium hydrogen sulfate 5 mM in methanol was used in the final method as polar modifier for the simultaneous determination of possible degradation products within 12 min. The present method can separate and detect related compounds such as isosorbide-2,5-dinitrate, isomannidemononitrate and isosorbide-2-mononitrate at the 0.1% (w/w) level as required by regulatory guidelines. Nitrate can be detected down to about 0.02% (w/w). Repeated analyses of ground tablet powder gave an assay precision for isosorbide-5-mononitrate of 1.4% (R.S.D., eight samples and two injections of each). For related substances at an area percent of 0.1 the precision was less than 10%.

Title: Elution of cationic species

with/without ion pair reagents from polar stationary phases via supercritical fluid chromatography

Authors: Zheng J, Taylor LT, Pinkston JD

Journal: Chromatographia 2006 Address: Virginia Tech,

Department of Chemistry, Blacksburg VA 24061-0212, USA.

Abstract: Traditionally, the application of supercritical fluid chromatography has been limited to compounds of relatively low polarity. Thus, we report here that one secondary amine and two quaternary amine salts were successfully eluted from a Deltabond Cyano-bonded silica column with the addition of sodium alkylsulfonate to the methanol-modified, carbon dioxide-based mobile phase. A possible ion-pairing interaction between the positively charged analytes and the anionic part of the sulfonate additive has been proposed. In another set of experiments, the three amine salts readily eluted from both Ethyl-pyridine-bonded silica and Amino-bonded silica phases without the need of additive although the peak shapes were less than desirable. The addition of sulfonate salt to the mobile

phase again sharpened the peaks. In the presence of a mixture of methanol and CO2, we suggest that these stationary phases are positively charged. We describe here also for the first time the employment of a strong silica-based anion exchange (SAX) column for supercritical fluid separation of cationic species. Two elution mechanisms were proposed for the three amine salts depending upon whether the ionic additive was incorporated into the mobile phase. Separation as the ion-pair was proposed with an ionic additive in the mobile phase; whereas separation of the intact amine salt from the positively charged basic stationary phases was suggested to be operational without the ionic additive.

Title: Feasibility of supercritical fluid

chromatography/mass spectrometry of polypeptides with up to 40-Mers

Authors: Zheng J, Pinkston JD, Zoutendam PH, Taylor LT

Journal: Anal Chem 2006 Address: Virginia Tech

Department of Chemistry, Blacksburg, Virginia 24061-0212, USA.

Abstract: Supercritical fluid chromatography (SFC) provides a number of advantages over traditional HPLC such as speed, practical use of longer columns, a normal-phase retention mechanism, and reduced use of organic solvents. Yet, it has been a technique traditionally limited to relatively nonpolar compounds. The nature of SFC mobile and stationary phases did not allow the elution of ionic compounds or of peptides, except, in the latter case, for the most hydrophobic peptides. The characterization of peptides is critically important for drug discovery and development in the pharmaceutical industry, as well as for a variety of other important applications. Here, for the first time to our knowledge, we show that relatively large peptides (at least 40 mers), containing a variety of acidic and basic residues, can be eluted in SFC. We used trifluoroacetic acid as additive in a CO2/methanol mobile phase to suppress deprotonation of peptide carboxylic acid groups and to protonate peptide amino groups. A 2-ethylpyridine bonded silica column, which was specifically developed for SFC, was used for the majority of this work. The relatively simple mobile phase was compatible with mass spectrometric detection.

Title: Comparative study of the

enantioselective separation of several antiulcer drugs by high-performance liquid chromatography and supercritical

Abstract: A comparative study of the enantiomeric separation of several antiulcer drugs such as omeprazole, lansoprazole, rabeprazole and pantoprazole using HPLC and supercritical fluid chromatography (SFC) on the Chrialpak AD column is presented in this work. The results show that employing the above mentioned column only two compounds (omeprazole and pantoprazole) could be

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fluid chromatography. Authors: Toribio L, del Nozal MJ, Bernal JL,

Alonso C, Jimenez JJ Journal: Journal Chromatogr. A. 2005

1091(1-2):118-123 Address: University of Valladolid,

Department of Analytical Chemistry, Faculty of Science, Prado de la Magdalena s/n, 47005 Valladolid, Spain.

enantiomerically resolved using HPLC, on the contrary SFC allowed the enantiomeric separation of all the compounds studied with higher resolutions and lower analysis times.

Title: Supercritical fluid

chromatography-tandem mass spectrometry for the enantioselective determination of propranolol and pindolol in mouse blood by serial sampling

Authors: Chen J, Hsieh Y, Cook J, Morrison R, Korfmacher WA

Journal: Anal Chem 2005 Address: Schering-Plough Research Institute,

Drug Metabolism and Pharmacokinetics Department, Kenilworth, New Jersey 07033

Abstract: Packed-column supercritical fluid chromatography (pSFC) coupled to an atmospheric pressure chemical ionization (APCI) source and a tandem mass spectrometer (MS/MS) with minimum sample pretreatment was explored for the rapid and enantioselective determination of (R,S)-propranolol in mouse blood. Serial bleeding of mice is advantageous for the reduction of animal usage, dosing errors, and animal-to-animal variation. The effects of the eluent flow rate and composition as well as the nebulizer temperatures on the ionization efficiency of racemic propranolol and pindolol as model compounds in the positive ion mode under pSFC conditions were studied. The fundamental parameters on the proposed hyphenated system such as matrix ionization suppression and chromatographic performances were investigated in improving sensitivity and enantiomeric separation for the detection of the analytes. The proposed chiral pSFC-APCI/MS/MS approach requiring 3 min/sample for the determination of (R,S)-propranolol at a low nanogram per milliliter region was partially validated with respect to specificity, linearity, reproducibility, and accuracy and was applied to support a pharmacokinetic study.

Title: Chromatography with two mobile

phases Authors: Wang M, Hou S, Parcher JF

Journal: Anal Chem 2005

Abstract: Experimental results for the investigation of chromatographic columns containing two mobile phases are presented. The eluent was composed of mixtures of methanol and carbon dioxide. The column was an uncoated fusedsilica- lined stainless steel capillary column. At certain experimental

Address: University of Mississippi, Chemistry Department, Mississippi 38677, USA

conditions, the eluent divided into two phases, both of which moved through the column. The predominant component of the liquid phase was methanol whereas the gas phase was composed of at least 93 mol % CO2. The columns were studied over a range of feed compositions (45-95 mol % CO2), pressures (61-101 bar), and temperatures (30-100°C). The compositions and densities of each phase were calculated from the Peng-Robinson equation of state. The residence times of the two mobile phases were determined by tracer pulse chromatography. The partition coefficients of a probe solute, benzene, were measured along with the retention times of neon and the total volume of the chromatographic column as a function of temperature, pressure, and stoichiometric feed composition. The calculated column volumes, that is the volume of the liquid and gas, were constant over the full range of feed composition. The partition coefficient of benzene was constant at fixed pressure and temperature, varied logarithmically with density at fixed temperature and feed composition, and displayed a maximum at intermediate temperatures at fixed pressure and feed composition. The measured retention times of neon were consistently equivalent to the calculated residence times of the gas phase, indicating that neon did not dissolve in the liquid phase and could thus serve as an accurate dead time marker. The implementation of chromatography with two mobile phases produces a chromatographic “window”. There is a lower limit for the retention volume of all solutes, viz., the residence time of the gas phase, exactly the same as normal chromatography. However, elimination of the stationary phase produces an upper limit to the retention volumes of solutes. This upper limit is the residence time of the liquid phase, so there is a retention window such that tG e ti e tL for all solutes.

Title: Universal acoustic flame detection

for modified supercritial fluid chromatography.

Authors: Xia Z, Thurbide KB

Journal: Journal Chromatogr. A. 2005 Nov 30

Address: University of Calgary, Department of Chemistry, 2500 University Dr NW, Calgary,

Abstract: A novel detector, based on the frequency of acoustic emissions from an oscillating premixed hydrogen/oxygen flame, has been characterized for use in supercritical-fluid chromatography (SFC). When an organic analyte is introduced, the steady pitch of the acoustic flame detector (AFD) increases proportionally to the carbon content of the molecule. Using standard hydrocarbon analytes, the SFC-AFD system provided a linear response over about 3 orders of magnitude with a detection limit (S/sigma=3) of 18ng of carbon per second. The detector sensitivity was uniform for all analytes and did not change when using either pure or methanol modified supercritical-carbon dioxide (SC-CO(2)) as a mobile phase. While a stable baseline could be obtained for a variety of

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Alta., Canada T2N 1N4. constant conditions, density gradients did cause it to shift due to the changing flow rate encountered when using a passive restrictor. While these changes were small for a pure SC-CO(2) mobile phase, they were larger when using a methanol modifier. Qualitatively, the AFD response compared well to a flame ionization detector (FID).

Title: Comparison of titania, zirconia,

and silica stationary phases for separating diesel fuels according to hydrocarbon group-type by supercritical fluid chromatography.

Authors: Paproski RE, Cooley J, Lucy CA

Journal: Journal Chromatogr. A. 2005 1095(1-2) 156-163.

Address: University of Alberta, Department of Chemistry, Gunning/Lemieux Chemistry Centre, Edmonton, Canada

Abstract: Silica, zirconia, and titania columns were compared for their ability to separate diesel samples into saturates, mono-, di-, tri-, and polyaromatics by supercritical fluid chromatography (SFC) using pure CO(2) according to ASTM method D 5186-03. A titania column coupled in series to a silica column was found to provide the highest overall group-type resolutions based on 20 model compounds, with resolutions as high as 14.7 for saturates versus monoaromatics and 11.9 for monoaromatics versus diaromatics. An oilsands-derived Synfuel light diesel, a commercial Ontario diesel, and a heavy Shell Canada Ltd. diesel blending feedstock were studied on a titania-silica coupled column as well as on a conventional silica column. The Synfuel results were similar (within 0.5 mass%) on both the conventional bare silica column and the titania-silica coupled column. The heavier commercial diesel and diesel blending feedstock samples yielded different results on the titania-silica coupled column compared to the silica column alone, demonstrating the importance of achieving the highest possible resolutions when baseline separation of group-types is not obtained.

Title: Molecularly imprinted polymers

for the enantioseparation of chiral drugs.

Authors: Ansell RJ

Journal: Adv Drug Deliv Rev. 2005 57(12) 1809-1835.

Address: University of Leeds, School of Chemistry, Leeds LS2 9JT, UK

Abstract: Molecularly imprinted polymers have been applied for many years in chiral separations and increasingly have been applied to drugs. Separation speed and efficiency in methods such as HPLC and CEC are increasingly competitive with alternative established CSPs. The literature on separation of drug enantiomers using MIPs via HPLC, TLC, SFC and CEC are reviewed. Issues for method development, particularly the monomer-template interactions during the imprinting process, and the mechanism of solute retention on MIP CSPs in chromatography using normal phase or reverse-phase mobile phases are considered.

Title: Preparative separation and

identification of derivatized beta-methylphenylalanine enantiomers by chiral SFC, HPLC and NMR for development of new peptide ligand mimetics in drug discovery.

Authors: Nogle LM, Mann CW, Watts WL Jr, Zhang Y

Journal: J Pharm Biomed Anal. 2005 Oct 17

Address: Wyeth Research, Discovery Analytical Chemistry, Chemical and Screening Sciences 500 Arcola Road, Collegeville, PA 19426, USA

Abstract: A direct preparative purification of all four isomers of the unnatural amino acid beta-methylphenylalanine was achieved using supercritical fluid chromatography (SFC) with stacked-injection. Final purification of the Cbz-methyl ester derived isomers was performed on a Daicel Chiralpak AD-H column (20mmx250mm), using 50:50 methanol/ethanol as the organic modifier and resulted in purification of over 3.4g of material in 6.25h with >90% total recovery. The absolute stereochemical assignment of the purified amino acids was determined through a combination of chiral HPLC, NMR and optical rotation studies. To our knowledge, this is the first reported preparative approach that has yielded all four compounds in a single chromatographic run.

Title: Enhanced chromatographic

resolution of amine enantiomers as carbobenzyloxy derivatives in high-performance liquid chromatography and supercritical fluid chromatography.

Authors: Kraml CM, Zhou D, Byrne N, McConnell O

Journal: Journal Chromatogr. A. 2005 1100(1) 108-115

Address: Wyeth Research, Discovery Analytical Chemistry,

Abstract: The carbobenzyloxy (cbz) protecting group is evaluated for it's potential to enhance the resolution of chiral amine enantiomers using high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). A series of cbz derivatives of commercially available racemates was prepared and analyzed by enantioselective chromatography using a variety of mobile phases and polysaccharide and Pirkle-type chiral stationary phases (CSPs). The cbz-derivatized product consistently demonstrated enhanced chiral resolution under HPLC and SFC conditions. Improved selectivity and resolution combined with an automated preparative HPLC or SFC system can lead to the rapid generation of highly purified enantiomers of desirable starting materials, intermediates or final products.

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CN 8000, 865 Ridge Road, Princeton, NJ 08543, USA.

Title: A simple subcritical

chromatographic test for an extended ODS high performance liquid chromatography column classification.

Authors: Lesellier E, Tchapla A


Address: LETIAM IUT d'Orsay EA 3343, Groupe de Chimie Analytique de Paris Sud, Plateau du Moulon, F-91400 Orsay, France.

Abstract: This paper describes a new test designed in subcritical fluid chromatography (SFC) to compare the commercial C18 stationary phase properties. This test provides, from a single analysis of carotenoid pigments, the absolute hydrophobicity, the silanol activity and the steric separation factor of the ODS stationary phases. Both the choice of the analytical conditions and the validation of the information obtained from the chromatographic measurements are detailed. Correlations of the carotenoid test results with results obtained from other tests (Tanaka, Engelhard, Sander and Wise) performed both in SFC and HPLC are discussed. Two separation factors, calculated from the retention of carotenoid pigments used as probe, allowed to draw a first classification diagram. Columns, which present identical chromatographic behaviors are located in the same area on this diagram. This location can be related to the stationary phase properties: endcapping treatments, bonding density, linkage functionality, specific area or silica pore diameter. From the first classification, eight groups of columns are distinguished. One group of polymer coated silica, three groups of polymeric octadecyl phases, depending on the pore size and the endcapping treatment, and four groups of monomeric stationary phases. An additional classification of the four monomeric groups allows the comparison of these stationary phases inside each group by using the total hydrophobicity. One hundred and twenty-nine columns were analysed by this simple and rapid test, which allows a comparison of columns with the aim of helping along their choice in HPLC.

Title: Screening approach for chiral

separation of pharmaceuticals. Part III. Supercritical fluid chromatography for analysis and purification in drug discovery.

Authors: Maftouh M, Granier-Loyaux C, Chavana E, Marini J, Pradines A, Heyden YV, Picard C

Abstract: High-throughput and performance analysis and purification of enantiomers are important parts of drug discovery and provide high-quality compounds for pharmacological testing. We have previously reported two parts describing chiral chromatographic screens using normal-phase (NPLC) and reversed-phase (RPLC) liquid chromatography, in order to cope with increasing numbers of new compounds generated by chemistry programs. We present in this part the development and implementation of a third faster screen using supercritical fluid chromatography

Journal: Journal Chromatogr. A. 2005 1088(1-2) 67-81

Address: Sanofi-Aventis Discovery Research Discovery Analytics, 195 Route d'Espagne, 31036 Toulouse, France

(SFC) to maximize chance in achieving rapid enantiomer resolution of large numbers of compounds in a minimum of time. The SFC screen utilizes a narrow combination of only four columns (Chirlapak AD and AS, and Chiralcel OD and OJ) and two solvent modifiers (methanol and isopropanol). A modifier and column-switching setup was employed to allow the entire screening process to be serially run in the order AD> OD > OJ > AS and methanol > isopropanol, so that the screening for a given molecule can be stopped when separation is achieved. The switching system was fully automated for unattended operation of multiple compounds. An optimization procedure was also defined, which can be performed if needed for unsuccessful separations in the screening step. The chiral SFC strategy proved its performance and robustness in resolution of hundreds proprietary chiral molecules generated by drug discovery programs, with a success rate exceeding 95%. In addition, the generic capability of the strategy was evaluated by applying the screen and optimization methodology to a test set comprising 40 marketed drugs differing from proprietary compounds in terms of chemical diversity, revealing a similar high success rate of 98%. Chiral separations developed at the analytical scale work easily and equally well at the semi-preparative level, as illustrated with an example. The SFC screen allows resolution of compounds that were partially separated by NPLC or not separated at all by RPLC, demonstrating the utility of implementing complementary chromatographic techniques. The SFC screen is currently an integral part of our analytical support to discovery chemical programs and is considered the first try for chiral separations of new compounds, because it offers a higher success rate, performance and throughput.

Title: Effect of system variables involved

in packed column SFC of nevirapine as model analyte using response surface methodology: application to retention thermodynamics, solute transfer kinetic study and binary diffusion coefficient determination.

Abstract: A multifactor optimization technique is successfully applied to study the effect of simultaneously varying the system variables on feasibility of nevirapine analysis by packed column supercritical fluid chromatography (PC-SFC). The optimal conditions were determined with the aid of the response surface methodology using 3(3) factorial designs. The method is based on methanol-modified carbon dioxide as the mobile phase at flow rate of 3.0 ml/min with elution through a JASCO Finepak SIL-5, [C18 (5-micron, 25 cm x 4.6 mm, i.d.)] column using photodiode array detection. The method has been successfully used to analyze commercial solid dosage form to

15

Authors: Kaul N, Agrawal H, Paradkar AR, Mahadik KR

Journal: J Biochem Biophys Methods. 2005 64(2) 121-141

Address: Bharati Vidyapeeth Deemed University Department of Quality Assurance Techniques, Poona College of Pharmacy, Erandwane, Pune, Maharashtra State, India.

assess the chromatographic performance of SFC system. The present work briefs the thermodynamic applications of PC-SFC with an emphasis on the results of nevirapine. The foremost of such applications is the determination of solute diffusion coefficient in supercritical mobile phase by Taylor-Aris peak broadening technique.

Title: Advantages and drawbacks of

popular supercritical fluid chromatography/mass interfacing approaches - a user's perspective.

Authors: Pinkston JD

Journal: Eur J Mass Spectrom 2005 11(2) 189-197.

Address: Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Rd, PO Box 8006, Mason, OH 45040, USA.

Abstract: Mobile phases in supercritical fluid chromatography (SFC) have low viscosities and high diffusion coefficients with respect to those of traditional high performance liquid chromatography (HPLC). These properties allow higher mobile phase flow rates and/or longer columns in SFC, resulting in rapid analyses and high efficiency separations. In addition, chiral SFC is becoming especially popular. Mass spectrometry (MS) is arguably the most popular "informative" detector for chromatographic separations. Most SFC/MS is performed with atmospheric pressure ionization (API) sources. Unlike LC/MS, the interface between the SFC column and the API source must allow control of the downstream (post-column) pressure while also providing good chromatographic fidelity. Here we compare and contrast the popular interfacing approaches. Some are simple, such as direct effluent introduction with no active back-pressure-regulator (BPR) in high speed bioanalytical applications. The pressure-regulating-fluid interface is more versatile and provides excellent chromatographic fidelity, but is less user friendly. The pre-BPR- split interface and an interface which provides total-flow-introduction with a mechanical BPR are good compromises between user friendliness and performance, and have become the most popular among practitioners. Applications of SFC/MS using these various interfaces are also discussed.

Title: Effect of ionic additives on the Abstract:

elution of sodium aryl sulfonates in supercritical fluid chromatography.

Authors: Zheng J, Taylor LT, Pinkston JD, Mangels ML


Address: Virginia Polytechnic Inst. and State Department of Chemistry, University College of Science, 107 Davidson Hall, Virginia Tech Blacksburg, VA 24061-0212, USA.

Addition of a small amount of polar solvent (i.e., modifier) to CO2 in packed column supercritical fluid chromatography (SFC) has shown major improvements in both polar analyte solubility and interaction of the polar analyte with the stationary phase. Recently, the addition of an ionic component (i.e., additive) to the primary modifier by one of us has been shown to extend even further the application of SFC to polar analytes. In this work, the effect of various ionic additives on the elution of ionic compounds, such as sodium 4-dodecylbenzene sulfonate and sodium 4-octylbenene sulfonate, has been studied. The additives were lithium acetate, ammonium acetate, tetramethylammonium acetate, tetrabutylammonium acetate, and ammonium chloride dissolved in methanol. Three stationary phases with different degrees of deactivation were considered: conventional cyanopropyl, deltabond cyanopropyl, and bare silica. The effect of additive concentration and additive functionality on analyte retention was investigated. Sodium 4-dodecylbenzene sulfonate was successfully eluted using all the additives with good peak shape under isocratic/isobaric/isothermal conditions. Different additives, however, yielded different retention times and in some cases different peak shapes.

Title: Application of supercritical fluid

chromatography in the quantitative analysis of minor components (carotenes, vitamin E, sterols, and squalene) from palm oil.

Authors: Choo YM, Ng MH, Ma AN, Chuah CH, Hashim MA

Journal: Lipids. 2005 40(4) 429-32

Address: Persiaran Institusi Malaysian Palm Oil Board (MPOB), No. 6, Bandar Baru Bangi, 43000 Kajang, Selangor, Malaysia

Abstract: The application of supercritical fluid chromatography (SFC) coupled with a UV variable-wavelength detector to isolate the minor components (carotenes, vitamin E, sterols, and squalene) in crude palm oil (CPO) and the residual oil from palm-pressed fiber is reported. SFC is a good technique for the isolation and analysis of these compounds from the sources mentioned. The carotenes, vitamin E, sterols, and squalene were isolated in less than 20 min. The individual vitamin E isomers present in palm oil were also isolated into their respective components, alpha-tocopherol, alpha-tocotrienol, gamma-tocopherol, gamma-tocotrienol, and delta-tocotrienol. Calibration of all the minor components of palm as well as the individual components of palm vitamin E was carried out and was found to be comparable to those analyzed by other established analytical methods.

17

Title: Enantioseparation of 1-phenyl-1-

propanol on chiralcel OD by supercritical fluid chromatography. I. Linear isotherm.

Authors: Rajendran A, Mazzotti M, Morbidelli M


Address: Institute of Process Engineering ETH Swiss Federal Institute of Technology Zurich, Sonneggstrasse 3, CH-8092 Zurich, Switzerland

Abstract: The supercritical fluid chromatography (SFC) separation of the enantiomers of 1-phenyl-1-propanol on the chiral stationary phase Chiralcel OD under linear conditions is studied. Supercritical CO2 modified with methanol is used as a mobile phase. The effect of modifier concentration, pressure and temperature is studied. An empirical isotherm to account for the effect of density of the mobile phase and modifier concentration has been used to model the experimental results. It was observed that the selectivity and resolution were higher at 30 degrees C as compared to those at 40oC.

Title: Integration of supercritical fluid

chromatography into drug discovery as a routine support tool. II. investigation and evaluation of supercritical fluid chromatography for achiral batch purification.

Authors: White C, Burnett J

Journal: Journal Chromatogr. A. 2005 1074(1-2) 175-185.

Address: Eli Lilly and Company Limited, Lilly Research Centre, Analytical Technologies,

Abstract: Supercritical fluid chromatography (SFC) has recently been implemented within our analytical technologies department as a purity assessment and purification tool to complement HPLC for isomer and chiral separations. This report extends the previous work to achiral analysis and purification. This internal evaluation explores the potential impact SFC can have on high throughput, batch purification. Achiral methods have been optimised and batches of compounds purified using a retention time mapping strategy. Here the preparative retention time is predicted from a standard calibration curve and fraction windows set to ensure the peak of interest is collected in one of the four available fraction positions. In this contribution, a completely indirect scale up strategy is applied using totally independent analytical and preparative methods. This novel approach allows for fast analytical purity analysis without compromising the ability to scale up to the preparative system. The benefits and limitations of SFC for batch purification are described in comparison to HPLC across a set of standard compounds and a set of 90 research compounds.

Erl Wood Manor, Sunninghill Road, Windlesham, Surrey GU206PH, UK

Title: Integration of supercritical fluid

chromatography into drug discovery as a routine support tool. Part I. fast chiral screening and purification.

Authors: White C


Address: Eli Lilly and Company Limited, Lilly Research Centre, Analytical Technologies, Erl Wood Manor, Sunninghill Road, Windlesham, Surrey GU206PH, UK

Abstract: Supercritical fluid chromatography (SFC) has been implemented within our group as a purity assessment and purification tool to complement high performance liquid chromatography (HPLC) for diastereomer and chiral separations. Using a novel strategy, rapid chiral screening has been implemented using short columns, high flow rates and fast gradients. A primary screen delivers a separation assessment using one solvent modifier (methanol) and four columns (Chiralpak AD-H and AS-H, and Chiralcel OD-H and OJ-H) run serially in a total of 24 min. A secondary screen then uses ethanol and isopropanol (IPA) modifiers across the same columns. The screens can be combined to run a sequence of samples overnight where each racemate is analysed over 80 min. The fast analytical screening and optimisation process enables rapid identification of the purification method. Furthermore, subsequent preparative chiral SFC has decreased the overall sample turnaround time for the Medicinal Chemist, delivering high fraction purities and acceptable recoveries, substantial operational cost savings and increased flexibility with respect to large scale purification feasibility in comparison to HPLC. SFC has been so successful it is now used as the primary method for chiral analysis and purification within our laboratory.

Title: Supercritical fluid chromatography

tandem-column method development in pharmaceutical sciences for a mixture of four stereoisomers.

Authors: Barnhart WW, Gahm KH, Thomas S, Notari S, Semin D, Cheetham J

Journal: J Sep Sci. 2005 28(7) 619-626

Address: Amgen Inc.

Abstract: A tandem-column method using Chiralpak AD-H and Chiralcel OD-H columns was achieved for baseline separation of a mixture of chiral pharmaceutical compounds (i.e., four stereoisomers) via supercritical fluid chromatography (SFC) with a mobile phase consisting of 90% liquid carbon dioxide and 10% ethanol:isopropanol (50:50 v/v). On the contrary, this mixture (mixture A) could not be baseline separated by SFC conditions explored with individual Chiralpak AD-H and Chiralcel OD-H columns. The effects of various mobile phases on elution order, capacity factor, selectivity, and resolution were determined with mixture A on the individual aforementioned columns to develop the tandem-column method.

19

Discovery Analytical Sciences, Molecular Structure, Thousand Oaks, CA 91320, USA

Title: Chiral separation of amines in

subcritical fluid chromatography using polysaccharide stationary phases and acidic additives.

Authors: Stringham RW


Address: Chiral Technologies, 730 Sprindale Drive, Exton, PA 19341, USA

Abstract: The chiral separation of basic compounds by subcritical fluid chromatography (SFC) is often unsuccessful, due possibly to multiple interactions of the analyte with the mobile and stationary phase. Incorporation of a strong acid, ethanesulfonic acid (ESA), into the sample diluent and mobile phase modifier gives a dramatic improvement in these separations. Screening with ethanol containing 0.1% ESA on CHIRALPAK AD-H gave separation of 36 of 45 basic compounds previously not separated in SFC. The mechanism appears to involve the separation of an intact salt pair formed between the basic compound and ESA. Other modifiers, other acids and one additional stationary phase were examined and found to yield additional separations.

Title: Reversal of elution order for

profen acid enantiomers in packed-column SFC on Chiralpak AD.

Authors: Gyllenhaal O, Stefansson M

Journal: Chirality 2005 17(5) 257-265.

Address: AstraZeneca R & D Analytical Development, Pharmaceutical and Analytical R & D, Molndal, Sweden

Abstract: Enantiomeric separations of four 2-substituted propionic acid drugs have been studied using packed-column supercritical fluid chromatography (SFC) with amylose tris(3,5-dimethylphenylcarbamate) coated on silica as support (Chiralpak AD). Under standard conditions (i.e., flow rate, 1.5 ml/min; column temperature, 30oC; back-pressure, 150 bar), the order of elution could be reversed when the polar alcohol modifier methanol in carbon dioxide was replaced by 2-propanol for ibuprofen, ketoprofen, and naproxen. For flurbiprofen, with the highest selectivity factor, no reversal was observed, although selectivity was reduced significantly with higher alcohols. Naproxen and flurbiprofen were also investigated with 2-butanol and 2-pentanol. The former showed reversal of elution order but not the latter. For higher alcohol modifiers, including 2-propanol, the peak symmetry was poor but could be improved by addition of citric acid in the alcohol modifier. These results stress the importance to investigate enantiomer elution order during the development of enantioselective methods and when chromatographic conditions are optimized. Preliminary experiments with column temperatures over the range of -15 to 45°C revealed that, in a

few cases, reversal took place with a change in temperature only.

Title: Enantioselective chromatography in drug discovery

Authors: Zhang Y, Wu DR, Wang-Iverson DB, Tymiak AA

Journal: Drug Discovery Today 2005 10(8) 571-577

Address: Bristol-Myers Squibb Company Discovery Analytical Sciences, Pharmaceutical Research Institute, P. O. Box 4000, Princeton, NJ 08648-4000, USA

Abstract: Molecular chirality is a fundamental consideration in drug discovery, one necessary to understand and describe biological targets as well as to design effective pharmaceutical agents. Enantioselective chromatography has played an increasing role not only as an analytical tool for chiral analyses, but also as a preparative technique to obtain pure enantiomers from racemates quickly from a wide diversity of chemical structures. Different enantioselective chromatography techniques are reviewed here, with particular emphasis on the most widespread high performance liquid chromatography (HPLC) and the rapidly emerging supercritical fluid chromatography (SFC) techniques. This review focuses on the dramatic advances in the chiral stationary phases (CSPs) that have made HPLC and SFC indispensable techniques for drug discovery today. In addition, screening strategies for rapid method development and considerations for laboratory-scale preparative separation are discussed and recent achievements are highlighted

Title: Isolation of palm tocols using

supercritical fluid chromatography.

Authors: Han NM, May CY, Ngan MA, Hock CC, Ali Hashim M

Journal: Journal Chromatogr. Sci. 2004 42(10) 536-539

Address: Persiaran Institusi, Malaysian Palm Oil Board, Bandar Baru Bangi, 43000 Kajang, Selangor, Malaysia.

Abstract: Crude palm oil contains 600 to 1000 ppm of tocols in the form of tocopherols and tocotrienols. These palm tocols have been isolated and analyzed in the past by various chromatographic techniques such as open column chromatography, high-performance liquid chromatography, as well as thin-layer chromatography. Supercritical fluid chromatography (SFC) has emerged as a more advanced chromatographic technique in recent years. The tocols present in palm oil are successfully isolated using SFC. Identification of these tocols is supported by various spectroscopic techniques such as 1H NMR, 13C NMR, and mass spectrometry.

Title: Enantioselective separations by

packed column subcritical and Abstract: Enantioselective separations have been one of the most successful applications of supercritical fluid

21


Authors: Phinney KW

Journal: Anal Bioanal Chem. 2005 382(3) 639-645

Address: National Institute of Standards and Technology, Analytical Chemistry Division, Chemical Science and Technology Laboratory, Gaithersburg, MD 20899-8392, USA.

chromatography (SFC). Although analytical scale separations have dominated the literature, the use of SFC for preparative chiral separations is growing. Both analytical and preparative scale SFC separations seek to take advantage of the high efficiency, high throughput, and rapid method development associated with the technique. This review will cover recent developments in the application of SFC to enantioseparations.

Title: Impacts of extraction methods in

the rapid determination of atrazine residues in foods using supercritical fluid chromatography and enzyme-linked immunosorbent assay: microwave solvent vs. supercritical fluid extractions.

Authors: El-Saeid MH, Kanu I, Anyanwu EC, Saleh MA

Journal: Scientific World Journal 2005 4(5) 11-19

Address: Texas Southern University, Department of Chemistry and Environmental Toxicology, 3100 Cleburne Avenue, Houston, Texas, USA.

Abstract: It is an accepted fact that many food products that we eat today have the possibility of being contaminated by various chemicals used from planting to processing. These chemicals have been shown to cause illnesses for which some concerned government agencies have instituted regulatory mechanisms to minimize the risks and the effects on humans. It is for these concerns that reliable and accurate rapid determination techniques are needed to effect proper regulatory standards for the protection of people's nutritional health. This paper, therefore, reports the comparative evaluation of the extraction methods in the determination of atrazine (commonly used in agricultural as a herbicide) residues in foods using supercritical fluid chromatography (SFC) and enzyme-linked immunosorbent assay (ELISA) techniques. Supercritical fluid extraction (SFE) and microwave solvent extraction (MSE) methods were used to test samples of frozen vegetables, fruit juice, and jam from local food markets in Houston. Results showed a high recovery percentage of atrazine residues using supercritical fluid coupled with ELISA and SFC than with MSE. Comparatively, however, atrazine was detected 90.9 and 54.5% using SFC and ELISA techniques, respectively. ELISA technique was, however, less time consuming, lower in cost, and more sensitive with low detection limit of atrazine residues than SFC technique.

Title: Separation of rosemary antioxidant compounds by supercritical fluid chromatography on coated packed capillary columns.

Authors: Ramirez P, Senorans FJ, Ibanez E, Reglero G

Journal: J Chromatogr A. 2004 1057(1-2) 241-245

Address: Universidad Autonoma de Madrid Area de Tecnologia de Alimentos, Facultad de Ciencias, Unidad Asociada al CSIC, 28049 Cantoblanco, Madrid, Spain.

Abstract: Antioxidant compounds in rosemary extracts obtained by supercritical fluid extraction (SFE) were separated by supercritical fluid chromatography (SFC) on packed capillary columns. The columns contained silica particles coated with SE-54 (5% phenyl, 95% methyl silicone) and Carbowax 20 M [poly(ethylene glycol)]. The use of coated packed capillary columns allowed the separation of polar compounds by SFC with neat CO2. The SFC conditions were selected on the basis of previous work. High pressures (up to 370 atm; 1 atm = 10,325 Pa) and moderate temperatures (up to 100 degrees C) were used to separate the compounds responsible for the antioxidant activity such as carnosic acid and camosol while lower pressures were sufficient to separate the compounds of the essential oil.

Title: Separation methods for

antibacterial and antirheumatism agents in plant medicines.

Authors: Wen D, Liu Y, Li W, Liu H

Journal: J Chromatogr B Analyt Technol Biomed Life Sci. 2004 812(1-2) 101-117.

Address: Peking University, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Beijing 100871, PR China.

Abstract: Traditional oriental medicines (TOM), with a very long history and many remarkable features, are very popular in Asian countries, especially in China, Japan and Korea. With the development of advanced analytical techniques, the modernization of traditional medicine has become a hot area in recent years and some herbal medicines have been increasingly accepted in western countries. Separation and determination of active components in various herbal medicines are considered to be critical for the modernization process. Antibacterial and antirheumatism agents are widely distributed in many medical plants and commonly used in clinical treatment. Therefore, the development of effective separation methods for the quality control of herbal medicines is absolutely important. In this article, the separation methods for the analysis of antibacterial and antirheumatism compounds in TOM were reviewed, including thin layer chromatography (TLC), gas chromatography (GC), supercritical fluid chromatography (SFC), high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and related hyphenation techniques. Sample preparation procedures and further development of these methods were also discussed.

23

Title: Increasing UV detection sensitivity

in the supercritical fluid chromatographic analysis of alcohol polyethers.

Authors: Hoffman BJ, Taylor LT, Rumbelow S, Pinkston JD


Address: Virginia Tech, Department of Chemistry, Blacksburg, VA 24061, USA.

Abstract: Alcohol ethoxylates (AEOs) that contain a wide distribution of oligomers pose a challenge for ultraviolet (UV) absorbance detection due to the fact that the AEOs absorb strongly only in the range of commercial UV detectors between 190 and 200 nm. Most mobile phase components, with the exceptions of water and carbon dioxide, also absorb in this region. Ethoxylated hexadecanol and octadecanol were derivatized with disilazane-chlorosilane mixtures for the formation of phenyl containing silylethers. Derivatized samples were analyzed by supercritical fluid chromatography (SFC) coupled with both electrospray ionization mass spectrometry and UV absorbance detection. An increase in the number of phenyl groups incorporated into the derivatives increased the number of oligomers observed by UV detection. An increase in the number of oligomers detected increased the calculated average molar ethoxylate values. The average molar oligomer values calculated by SFC-UV for these alcohols were consistent with the nominal reported values.

Title: Chiral separation of omeprazole

and several related benzimidazoles using supercritical fluid chromatography.

Authors: del Nozal MJ, Toribio L, Bernal JL, Alonso C, Jimenez JJ

Journal: J Sep Sci. 2004 27(12) 1023-1029.

Address: University of Valladolid, Department of Analytical Chemistry, Faculty of Science, Prado de la Magdalena s/n, 47005 Valladolid, Spain.

Abstract: A study of the enantiomeric separation of omeprazole and several related benzimidazoles, using supercritical fluid chromatography (SFC), on the amylose based column Chiralpak AD is presented in this work. The effect of the organic modifier as well as temperature on the retention and enantioresolution was investigated. Alcohol-type modifiers provided the best results, allowing the enantiomeric separation of all the compounds studied with resolutions that were in most cases higher than 2, and analysis times lower than 10 minutes. An investigation of the temperature effect revealed that the isoelution temperature was below the working temperature range in only two cases, and hence it was better to work at the highest temperature permitted.

Title: Determination of alcohol polyether Abstract:

average molar oligomer value/distribution via supercritical fluid chromatography coupled with UV and MS detection.

Authors: Hoffman BJ, Taylor LT, Rumbelow S, Goff L, Pinkston JD

Journal: J Chromatogr A. 2004 1043(2) 285-290

Address: Virginia Tech, Department of Chemistry, Blacksburg, VA 24061, USA.

Supercritical fluid chromatography (SFC) was used for the analysis of ethoxylated and propoxylated surfactants. Samples were derivatized to phenylated silyl ethers with a disilazane-chlorosilane mixture. Addition of a phenyl group to the surfactant allowed UV-absorbance detection of each oligomer. Acetonitrile and methanol were evaluated as mobile phase modifiers. Better peak shape was realized with methanol-modified CO2 on an octadecyl silica bonded phase than with acetonitrile-modified CO2. Peak assignments were made via SFC coupled with electrospray ionization-mass spectrometry (ESI-MS) in the positive ion mode. A sulfonamide-embedded alkyl stationary phase was also evaluated for separation of the derivatized samples. SFC-UV and SFC-ESI-MS data were jointly used for calculation of average molar oligomer values which were then compared to values calculated from 1H NMR data of non-derivatized samples. The derivatization or separation method using the sulfonamide embedded phase required no preliminary cleanup and yielded reproducible oligomer values that were consistent with those of the manufacturer's nominal values.

Title: Rapid, automated screening

method for enzymatic transformations using a robotic system and supercritical fluid chromatography.

Authors: Di L, McConnell OJ, Kerns EH, Sutherland AG

Journal: J Chromatogr B Analyt Technol Biomed Life Sci. 2004 809(2) 231-235

Address: Wyeth Research, P.O. Box CN 8000, Princeton, NJ 08543-8000, USA

Abstract: An automated screening method was developed for enzymatic transformations using a robotic system and rapid chiral supercritical fluid chromatography (SFC) analysis with a run time of 1.5 min. The method accelerates the enzyme selection process for screening biocatalysts, where a large number of enzymes are evaluated for activity and enantioselectivity. Kinetic resolution of secondary alcohols by enzymatic transesterification was used as a prototype for method development. The rapid automated method can be used effectively for screening enzymes and optimizing reaction conditions in biocatalysis.

Title: Chiral separation of some triazole Abstract:

25

pesticides by supercritical fluid chromatography

Authors: Toribio L, Nozal MJ, Bernal JL, Jimenez JJ, Alonso C

Journal: J Chromatogr A. 2004 1046 249-253

Address: University of Valladolid, Department of Analytical Chemistry, Faculty of Sciences, Prado de la Magdalena s/n, E-47005 Valladolid, Spain.

The enantiomeric separation of six triazole pesticides: cyproconazole, propiconazole, diniconazole, hexaconazole, tebuconazole and tetraconazole, using supercritical fluid chromatography and the Chiralpak AD column, is presented in this work. The effect of different organic modifiers such as methanol, ethanol and 2-propanol on the retention and resolution was studied as well as the presence of additives in the mobile phase. The results obtained were highly successful, all the compounds were enantiomerically separated and in most of the cases the analysis time was close to 10 min. The type of organic modifier that provided the best results depended on the compound.

Title: Effect of amine mobile phase

additives on chiral subcritical fluid chromatography using polysaccharide stationary phases.

Authors: Ye YK, Lynam KG, Stringham RW


Address: Chiral Technologies Inc. 730 Springdale Drive, Exton, PA 19341, USA.

Abstract: Increased retention and selectivity in the subcritical fluid chromatography (SFC) of various amine compounds on polysaccharide chiral stationary phases (CSP) was observed upon incorporation of cyclic amines into the modifier. The retention increases are most pronounced with 2-propanol and are almost absent when methanol is used as modifier. This suggests that the effect may arise from a restriction to the modifier access to the binding site required to effect elution. The effect of the amine additives in SFC does not remain after their removal from the mobile phase. Findings were applied to the development of a 5 min separation of amphetamine and methamphetamine enantiomers.

Title: Packed column supercritical fluid

chromatography of a peroxysome proliferator-activating receptor agonist drug. Achiral and chiral purity of substance, formulation

Abstract: This paper describes packed column supercritical fluid chromatography (SFC) for the analysis of a peroxysome proliferator-activating gamma-receptor agonist that is a carboxylic acid. Evaluation of conditions for the separation of this candidate drug and related compounds in bulk substance is described. A Chiralcel OD column was used for this purpose due to its high selective retention of related substances and relative inertness, though the enantioselectivity was negligible, with

assay and its enantiomeric purity. Authors: Gyllenhaal O

Journal: J Chromatogr A. 2004 1042(1-2) 173-180.

Address: AstraZeneca R & D Molndal, Analytical Development, S-431 83 Molndal, Sweden.

methanol as polar modifier. A high enantioselectivity was obtained on Chiralpak AD and it was possible to determine the enantiomeric purity within 10 min on a 5 cm short column. Both the achiral and the chiral systems were run without acid additive in the mobile phase and the level of detection of impurities by area was about 0.1%. For the analysis of samples dissolved in water, without any isolation step, 2-propanol was used as modifier. Due to the column surface activity, evidently generated by injected water, citric acid 1 mM was included as additive in the 2-propanol in order to maintain symmetric and undistorted peak shape. The detection limit for the assay was 21 microg mL(-1) (50 nmol mL(-1)) for 5 microL injected (R.S.D. 6.4%, n = 8). A 5 cm short Chiralcel OD column was used. Determination of enantiomeric purity of the drug in aqueous samples required increased sensitivity. The sample was acidified and extracted into a small volume of 1-pentanol, out of which 25 microl was analyzed by SFC. The minor enantiomer at the 3% (w/w) level added could be confirmed. Its ratio remained constant during the procedure as measured relative to a reference solution in organic media.

Title: Hold-and-flush, a novel fraction

collection method in semi-preparative subcritical and supercritical fluid chromatogra

Authors: Wu J, McAllister H


Address: Johnson and Johnson Pharmaceutical Research and Development, Drug Discovery, L.L.C., 3210 Merryfield Row, San Diego, CA 92121-1126, USA

Abstract: In supercritical fluid chromatography, the partial phase separation that occurs in the dead volume between automatic backpressure regulator and collection vessels causes significant peak tailing and delayed arrival of compounds to the collectors. As a result, when two peaks are barely baseline separated, it becomes very difficult to correctly set fraction collection triggers, which in turn results in fractions being collected with lower purity and lower yield. The problem can be solved with a simple addition of a four-port, two-position switching valve between UV detector and automatic back pressure regulator. The valve acts as a timed gate to release each peak out of the back pressure regulator and into the collection vessel, while at the same time it holds later peaks inside the UV detector and the column in a closed loop.

Title: A method for extraction and

quantification of ginkgo terpene Abstract: A method was developed for the extraction and quantification of pharmacologically active terpene

27

trilactones Authors: Ding C, Chen E, Zhou W, Lindsay RC

Journal: Anal Chem. 2004 76 4332-4336

Address: University of Wisconsins Department of Food Science, and Wisconsin Center for Space Automation and Robotics, Madison, Wisconsin 53711

trilactones (ginkgolides, bilobalide) from the tissues of Ginkgo biloba L. and pharmaceutical ginkgo products by RPHPLC, based on the theory of terpene trilactones ionization. Four ginkgolides (GA, GB, GC, GJ) and bilobalide (BB) from both the ginkgo leaves and commercially available ginkgo extracts were quantitatively extracted by using this method. The recovery rate of the method was 97.5-100% with RSD of 1.2-2.8%. The detection limit was 0.05-0.1 íg, and the linear range was 0.1-12 íg. This detection limit represents a marked improvement over previously reported methods, suggesting the new method is a viable technique for routine analysis of ginkgo terpene trilactones in natural and commercial samples. The method reported by van Beek et al. in 1991 (van Beek, T. A.; Scheeren, H. A.; Rantio T.; Melger, W. C.; Lelyveld, G. P. J. Chromatogr. 1991, 543, 375-387.) was used as a reference method to monitor the accuracy of extraction and analysis in this study. SSI-MS technique was used to identify isolated target components. Carbohydrase treatment and solubility of terpene trilactones in various solvents were also discussed.

Title: Comprehensive two-dimensional

supercritical fluid and gas chromatography with independent fast programmed heating of the gas chromatographic column.

Authors: Venter A, Rohwer ER

Journal: Anal Chem. 2004 76(13) 3699-3706.

Address: University of Pretoria, Chemistry Department, South Africa.

Abstract: With comprehensive two-dimensional supercritical fluid and fast, independent temperature-programmed gas chromatography (SFCxGC), a polar column was used in the first dimension to achieve group-type analysis. The eluent of this separation was repetitively sampled and transferred to a fast, resistively heated gas chromatograph to obtain the boiling point distribution over the entire polarity separation. The SFC was operated isothermally with stopped flow to provide a sufficient time span for the GC analysis. The GC analysis had a typical cycle time of 1 min for the system demonstrated here. During this time, the GC column was independently heated at a rate of 450 degrees C/min to 250 degrees C and actively cooled again to -50 degrees C before the next GC injection took place. The analysis of petrochemical samples is presented to illustrate the technique.

Title: Porous electrodes supported on

ion-exchange membranes as electrochemical detectors for supercritical fluid chromatography

Abstract: A conveniently assembled electrochemical cell, exploiting a porous electrode supported on a moist perfluorinated ion-exchange polymer, is proposed for profitable electrochemical detection in supercritical fluid chromatography. It consists of a porous Pt working electrode, contacted by the

Authors: Toniolo R, Comisso N, Schiavon G, Bontempelli G

Journal: Anal Chem. 2004 76(13) 3699-3706.

Address: University of Udine, Department of Chemical Sciences and Technology, Via Cotonificio 108, I-33100 Udine, Italy

mobile phase from the chromatographic column, which is chemically deposited onto one side of a Nafion membrane. The rear uncoated side of this membrane, acting as a solid polymer electrolyte, is contacted by an electrolyte solution (1 M NaCl) contained in an internal compartment equipped with a Pt counter electrode and a Ag/AgCl, Cl- 1Mreference electrode. Ferrocene, eluted with supercritical carbon dioxide through a Spherisorb column installed in a supercritical fluid chromatographic system, was used as electroactive prototype analyte to test the performance of this detector, which turned out to be quite better than that provided by a conventional on-line UV absorbance detector. The recorded peaks were characterized by both a good reproducibility (4.5%) and a linear dependence of their height and area, which extended over a wide concentration range ( 3 orders of magnitude). Moreover, they were not interfered by possible solvent front, unlike peaks recorded by the UV detector. The detection limit, estimated for a signal-tonoise ratio of 3 (4.2 10-11 mol), was lower by 1 order of magnitude than that found for the UV detector. Finally, the long-term stability of this detector was satisfactory in that only a 6% decrease in the current responses was observed after a rather long period (2 months) of continuous use.

Title: Determination of thermodynamic

properties by supercritical fluid chromatography.

Authors: Roth M


Address: Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, 61142 Brno, Czech Republic.

Abstract: This survey attempts to summarize thermodynamic applications of supercritical fluid chromatography (SFC) with an emphasis on the results published during the last 10 years. In addition to a review of thermodynamic measurements by SFC, it contains brief sections on instrumental considerations and on the sources of auxiliary information needed when processing the retention data.

Title: High-throughput preparative

process utilizing three complementary chromatographic

Abstract: A high-throughput process was developed in which wells in plates generated from parallel synthesis are automatically channeled to an appropriate purification technique using analytical data as a

29

purification technologies. Authors: Ventura M, Farrell W, Aurigemma C,

Tivel K, Greig M, Wheatley J, Yanovsky A, Milgram KE, Dalesandro D, DeGuzman R, Tran P, Nguyen L, Chung L, Gron O, Koch CA

Journal: J Chromatogr A. 2004 1036(1) 7-13.

Address: Pfizer Global R&D-La Jolla, 10770 Science Center Drive, San Diego, CA 92121, USA.

guide. Samples are directed to either of three fundamentally different preparative techniques: HPLC with UV-triggered fraction collection, supercritical fluid chromatography (SFC) with UV-triggered fraction collection, or HPLC with MS-triggered fraction collection. Automated analysis of the analytical data identifies the product compound mass and creates work lists based on chromatographic properties exhibited in the data so that each preparative instrument cherry picks the appropriate list of samples to purify when a preparative-scale plate is loaded.

Title: Separation of derivatized alcohol

ethoxylates and propoxylates by low temperature packed column supercritical fluid chromatography using ultraviolet absorbance detection.

Authors: Hoffman BJ, Taylor LT, Rumbelow S, Goff L, Pinkston JD


Address: Virginia Polytechnic Inst. and State, Department of Chemistry, University College of Arts and Sciences, 107 Davidson Hall, Blacksburg, VA 24061-0212, USA.

Abstract: Supercritical fluid chromatography (SFC) is capable of separating oligomers of alcohol ethoxylates (AEOs) and propoxylates (APOs) samples with pure carbon dioxide. The instrumental conditions, however, needed for separation necessitate both high temperature and high pressure. Derivatization of alcohol polyether samples with an UV absorbing agent has been achieved with a phenylated disilazane in hopes of employing a solvent-modified CO2 mobile phase in conjunction with both lower CO2 pressure and lower temperature for oligomer separation. A silylether containing a single phenyl group was formed via the derivatization of the hydroxyl termini of AEO and APO samples. The derivatized polyethers were detected at 215 nm with little or no interference from the mobile phase. Octadecylsilica (ODS) and a polar embedded alkyl bonded silica stationary phase were studied with the organic solvent-modified CO2 mobile phase. The combination of an ODS phase and the polar embedded phase, tandemly stacked, produced the best chromatographic separation of oligomeric species. Data from SFC-UV separations combined with peak assignments from SFC with electrospray ionization-mass spectrometric (ESI-MS) detection produced average molar oligomer values for each surfactant sample.

Title: The coupling of supercritical fluid Abstract:

chromatography and field ionization time-of-flight high-resolution mass spectrometry for rapid and quantitative analysis of petroleum middle distillates.

Authors: Qian K, Diehl JW, Dechert GJ, DiSanzo FP

Journal: Eur J Mass Spectrom (Chichester, Eng). 2004 10(2) 187-189

Address: ExxonMobil Research and Engineering Company, 1545 Route 22 East, Annandale, NJ 08502, USA.

We report the first coupling of supercritical fluid chromatography (SFC) with field ionization time-of-flight high-resolution mass spectrometry (FI-ToF HRMS), in parallel with ultraviolet (UV) detection and flame ionization detection (FID), for rapid and quantitative analysis of petroleum middle distillates. SFC separates petroleum middle distillates into saturates and 1- to 3-ring aromatics. FI generates molecular ions for hydrocarbon species eluted from the SFC. The high resolution and exact mass measurements by ToF mass spectrometry provide elemental compositions of the molecules in the petroleum product. The amounts of saturates and aromatic ring types were quantified using the parallel SFC-FID assisted by SFC-UV. With a proper carbon-number calibration, the detailed composition of the petroleum middle distillate was rapidly determined.

Title: Enantioseparation of

hydrochloride salts using carbon dioxide-based mobile phases with on-line polarimetric detection.

Authors: Geiser F, Shah R

Journal: Chirality 2004 16(4) 263-266

Address: Johnson Matthey Inc., Pharmaceutical Materials, West Deptford, New Jersey 08066, USA

Abstract: Most HPLC enantioseparations of amine analytes are performed using normal-phase systems containing mobile phases of heptane with ethanol (or 2-propanol) and an amine additive. Since salt-forms of amine analytes are usually insoluble in normal-phase eluents, free-base forms are synthesized for preparative chromatography. It would be highly desirable to directly chromatograph salt forms of amine analytes using mobile phases of carbon dioxide (CO(2)) and methanol (MeOH). Such separations would be readily suitable for preparative chromatography, since most amine salts are highly soluble in MeOH. In this article, advantages are shown for the use of supercritical fluid chromatography (SFC) instrumentation with tandem UV and polarimetric detection for confirming enantioseparation as well as for determining optimum preparative column injections. Examples are shown for racemic mixtures of propranolol HCl (I), thioridazine HCl (II), tramadol HCl (III), and flurbiprofen (IV), all of which resolved on Chiralpak AD-H chiral stationary phase using mobile-phase systems of CO(2) and MeOH without the use of basic or acidic additives.

31

Title: Comparison of preparative HPLC/MS and preparative SFC techniques for the high-throughput purification of compound libraries.

Authors: Searle PA, Glass KA, Hochlowski JE

Journal: J Comb Chem. 2004 6(2) 175-180.

Address: Abbott Laboratories, Medicinal Chemistry Technologies, Global Pharmaceutical Research & Development, Department R4CP, Building AP10, 100 Abbott Park Road, Abbott Park, IL 60064-6101, USA.

Abstract: A diverse set of 16 high-throughput organic synthesis libraries, consisting of 48 samples per library, has been purified by both preparative supercritical fluid chromatography (SFC) and preparative high-performance liquid chromatography (HPLC). This paper details the relative effectiveness of these two purification techniques in terms of success, yield, and purity of final product.

Title: Lipid-lowering effects of ethyl 2-

phenacyl-3-aryl-1H-pyrrole- 4-carboxylates in rodents

Authors: Holub JM, O'Toole-Colin K, Getzel A, Argenti A, Evans MA, Smith DC, Dalglish GA, Rifat S, Wilson DL, Taylor BM, Miott U, Burnham s

Journal: Molecules 2004 9 135-157

Address: Rider University, Department of Chemistry and Biochemistry, 2083 Lawrenceville Rd., Lawrenceville, NJ 08648, U.S.A.

Abstract: A series of substituted 2-phenacyl-3-phenyl-1H-pyrrole-4-carboxylates were prepared from substituted acetophenones in six steps. The final condensations between a chloroenal and an aminoketone were carried out under neutral conditions in parallel to yield the series listed below. Selected pyrrole derivatives proved to be potent hypolipidemic agents lowering serum triglyceride concentrations in CF-1 male mice after 14 days of I.P. administration. One agent orally lowered serum cholesterol in Sprague-Dawley male rats at 2mg/kg/day after 14 days. The agents demonstrated a lowering of mouse serum LDLcholesterol levels and selected compounds showed an elevation of serum HDL-cholesterol levels. The cholesterol concentrations in the liver were raised while the cholesterol and triglyceride contents of the aorta were significantly lowered by the selected trisubstituted pyrrole.

Title: Determination of the stoichiometry

of protein complexes using liquid chromatography with fluorescence and mass spectrometric detection of fluorescently labeled proteolytic peptides.

Authors: Hochleitner EO, Sondermann P, Lottspeich F

Journal: Proteomics. 2004 4(3) 669-676

Address: Max Planck Institute of Biochemistry, Analytical Protein Chemistry Group, Martinsried, Germany.

Abstract: A method for the determination of the stoichiometry of protein complexes has been developed, which is based on proteolytic digestion of the complex, labeling with a fluorescent reagent, specific for amino or sulfhydryl groups, and separation by liquid chromatography with fluorescence and mass spectrometric detection. The intensity of the fluorescence signal of the labeled peptides resulting from different proteins is directly proportional to the stoichiometry of these proteins in the complex. The performance of the method was evaluated with standard peptides and proteins to ensure that accurate molar ratios can be obtained from the fluorescence chromatogram. Standard deviations of the measured molar ratio from the expected molar ratio were below 10% for both peptides and proteins. The method was finally employed for the determination of the stoichiometry of the 1:1 complex of sFc gamma RIII and hFc1. Using the described methodology, a stoichiometry of 1:1.1 was measured, which agrees well with a 1:1 complex.

Title: Pesticide residues in canned

foods, fruits, and vegetables: the application of supercritical fluid extraction and chromatographic techniques in the analysis.

Authors: El-Saeid MH

Journal: ScientificWorldJournal. 2003 3 1314-1326.

Address: AL-Azhar University, Department of Food Science and Technology, Nasr City, Cairo, Egypt.

Abstract: Multiple pesticide residues have been observed in some samples of canned foods, frozen vegetables, and fruit jam, which put the health of the consumers at risk of adverse effects. It is quite apparent that such a state of affairs calls for the need of more accurate, cost-effective, and rapid analytical techniques capable of detecting the minimum concentrations of the multiple pesticide residues. The aims of this paper were first, to determine the effectiveness of the use of Supercritical Fluid Extraction (SFE) and Supercritical Fluid Chromatography (SFC) techniques in the analysis of the levels of pesticide residues in canned foods, vegetables, and fruits; and second, to contribute to the promotion of consumer safety by excluding pesticide residue contamination from markets. Fifteen different types of imported canned and frozen fruits and vegetables samples obtained from the Houston local food markets were investigated. The major types of pesticides tested were pyrethroids, herbicides, fungicides, and carbamates. By using these techniques, the overall data showed 60.82% of the food samples had no detection of any pesticide residues under this investigation. On the other hand, 39.15% different food samples werecontaminated by four different

33

pyrethroid residues +/- RSD% ranging from 0.03 +/- 0.005 to 0.05 +/- 0.03 ppm, of which most of the pyrethroid residues were detected in frozen vegetables and strawberry jam. Herbicide residues in test samples ranged from 0.03 +/- 0.005 to 0.8 +/- 0.01 ppm. Five different fungicides, ranging from 0.05 +/- 0.02 to 0.8 +/- 0.1 ppm, were found in five different frozen vegetable samples. Carbamate residues were not detected in 60% of investigated food samples. It was concluded that SFE and SFC techniques were accurate, reliable, less time consuming, and cost effective in the analysis of imported canned foods, fruits, and vegetables and are recommended for the monitoring of pesticide contaminations.

Title: Separation and trace estimation of

benzidine and its macromolecular adducts using supercritical fluid chromatography.

Authors: Patel G, Agrawal YK

Journal: J Chromatogr B Analyt Technol Biomed Life Sci. 2003 795(2) 157-165.

Address: Gujarat University, Department of Biochemistry, School of Sciences, Ahmedabad, Gujarat 380009, India

Abstract: A sensitive, rapid, selective and reproducible method has been developed to measure blood plasma levels of benzidine (BZ) and its acetylated metabolite, N-OH-N,N'-diacetylbenzidine (N-OH-DABZ), using supercritical fluid chromatography (SFC) for the first time. Benzidine and N-OH-N,N'-diacetylbenzidine were extracted from the plasma using ether. Separation was done on a Nucleosil (250 mm x 4.6 mm) 10 microm, Nucleosil-RP-C18 column with 7.4% (v/v) methanol-modified supercritical fluid carbon dioxide (2.5 ml min(-1)) as mobile phase. The column temperature was 45oC and the outlet pressure was set at 8.83 MPa. The detection was done using a UV-Vis detector set at 280 nm. The limit of quantification was 0.10 ng ml(-1) (BZ) and 0.14 ng ml(-1) (N-OH-diacetylbenzidine) using 1 ml plasma specimen. The mean extraction recovery of BZ was found to be 98.6%. The SFC method was directly compared to a published HPLC-UV method. With respect to speed, organic solvent usage, sensitivity, specificity and accuracy, SFC was found to be superior. The method has been successfully used to estimate the BZ, N-OH-diacetylbenzidine levels in blood plasma of the animals who were administered 15 microg kg(-1) body weight of benzidine.Further, this method has been also applied for the detection and quantification of benzidine DNA and hemoglobin adducts from the blood and tissue samples of the benzidine dosed animals.

Title: Advantages of the use of

monolithic stationary phases for modelling the retention in

Abstract: The low viscosity of supercritical fluids enables the coupling of columns, which favours both the high efficiency of separation and the ability of tuning the selectivity. However, it increases the inlet pressure then modifies the fluid density, i.e. the eluotropic strength of the mobile phase. In this case,

sub/supercritical chromatography Application to cis/trans - carotene separation

Authors: Lesellier E, West C, Tchapla A


Address: LETIAM, Plateau de Moulon, Orsay 91400, France

the latter is rather different depending on the number of coupled columns. This fact prevents the calculation of the chromatographic parameters for coupled columns from the results obtained from one. In subcritical conditions, by using silica rod columns, which have bimodal porous structure, the flow resistance parameter is dramatically reduced. Consequently, the addition of monolithic columns induces only slight internal pressure changes and the fluid density does not vary with the column length. In this case, the calculation of retention factor and selectivity based on retention values obtained on each separate column provides accurate results allowing to determine the optimum column length in regard to the studied separation. After a better characterisation of the stationary phase included in the Chromolith column, this paper describes the -carotene isomers separation obtained by coupling up to six Chromolith columns to an octadecyl bonded particulate one. These compounds were studied because of the difficulty to separate these cis/trans isomers. No abnormal apparent dead volume change due to fluid density variation was reported, and good correlations between experimental and calculated retention factors and selectivities were observed. The optimum separation requires five highly porous columns coupled to aYMCPack Pro. Moreover, the use of monolith packing allows to decrease both the retention factor and the analytical time by comparison to previous studies.

Title: Rapid method development for

chiral separation in drug discovery using sample pooling and supercritical fluid chromatography-mass spectrometry.

Authors: Zhao Y, Woo G, Thomas S, Semin D, Sandra P


Address: Amgen Inc., Discovery Analytical Science,

Abstract: A novel strategy for rapid chiral method development has been implemented using sample pooling and supercritical fluid chromatography-mass spectrometry (SFC-MS) on four chiral stationary phases, namely Chiralpak AD and AS, and Chiralcel OJ and OD, and eight different modifier concentrations (5 to 40% methanol-0.2% isopropylamine). The screening is performed under an outlet pressure of 110 bar at 35oC, and at a flow-rate of 2.5 ml/min for the initial 20 min and then ramped up to 4 ml/min and held for 4.5 min to elute all solutes from the column. The entire process is fully automated from injection to data processing, and operates unattended for 15 h overnight to obtain optimal chiral separation for multiple compounds. A unique feature of using SFC-MS to monitor chiral synthesis is the negligible interferences from achiral impurities. In addition, with SFC-MS, enantiomeric excess can be determined with much lower detection limits than UV and much shorter analysis times compared to normal-phase/reversed-phase liquid chromatography.

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One Amgen Center Drive, Mail Stop 29-M-B, Thousand Oaks, CA 91320, USA

Title: Use of semipreparative

supercritical fluid chromatography to obtain small quantities of the albendazole sulfoxide enantiomers.

Authors: Toribio L, Nozal MJ, Bernal JL, Nieto EM



Abstract: The semipreparative separation of the albendazole sulfoxide enantiomers using chiral supercritical fluid chromatography is presented in this work. For this purpose, a modular SFC chromatograph was adapted to work at semipreparative scale and a Chiralpak AD (250 x 10 mm) column was used. Different injection volumes were evaluated in order to obtain high purities and throughputs. Using the maximum load, it was possible to obtain 37 mg/h of the first eluted enantiomer with a purity of 99.9%, and 36.5 mg/h of the second eluted enantiomer with a purity of 95%.

Title: Isolation of ceramide fractions

from skin sample by subcritical chromatography with packed silica and evaporative light scattering detection

Authors: Lesellier E, Gaudin K, Chaminade P, Tchapla A, Baillet A


Address: Faculté de Pharmacie,

Abstract: Separative method of lipid classes from the stratum corneumwas developed with packed silica and supercritical CO2 containing 10% of methanol at 15oC, 15MPa and 3ml min−1. The elution order of lipid classes was first esterified cholesterol, triglycerides, squalene co-eluted in a single peak, then free fatty acids, free cholesterol, ceramides and finally glycosylceramides. The ceramides were eluted in several fractions which depended on the number of hydroxyl groups in the molecule, i.e. more hydroxyl groups were contained in ceramides, more important was the retention. Moreover, the retention was not altered by the presence of carbon double bond and variation of the alkyl chain length. The ceramide response with the evaporative light scattering detector was improved by turning the influence of the solvent nature on the response to advantage. Therefore, addition of various

Laboratoire de Chimie Analytique, rue J-B Clément, Chatenay-Malabry Cedex 92290, France LETIAM, Plateau de Moulon, Orsay 91400, France

solvents with or without triethylamine and formic acid were tested in post-column due to the incompatibility of such modifiers with silica stationary phase. Thereby the solvent conditions for the separation and the detection can be adjusted almost independently. The response was greatly increased by post-column addition of 1% (v/v) triethylamine and its equivalent amount of formic acid in dichloromethane introduced at 0.1 ml min−1 into the mobile phase. This device had allowed the detection of 400 ng of ceramide with a S/N = 21, whereas no peak was observed in absence of the post-column addition. Finally, the method was applied to the treatment of skin sample which led to highly enriched ceramide fraction.

Title: Preserving the chromatographic

integrity of high-speed supercritical fluid chromatography separations using time-of-flight mass spectrometry.

Authors: Bolanos BJ, Ventura MC, Greig MJ

Journal: J Comb Chem. 2003 5(4) 451-455

Address: Pfizer Global Research and Development, La Jolla Laboratories, 10770 Science Center Drive, San Diego, California 92121, USA.

Abstract: The advantage of high-speed time-of-flight-mass spectrometry (TOF-MS) detection for ultrafast qualitative supercritical fluid chromatography/mass spectrometry (SFC/MS) applications allows the superior resolving power of SFC to be exploited in high-throughput analysis. A chromatographic comparison of quadrupole MS and TOF-MS shows high-speed TOF total ion current data point sampling to be more indicative of fast SFC separations and corresponding short (1-2 s) baseline peak widths. Results shown for analysis of a six-compound mixture with two peaks eluting at 0.86 and 0.89 min exhibit >50% resolution by high-speed TOF data sampling, whereas the same peaks appear to coelute using quadrupole MS data sampling. Additionally, a marked improvement in the peak baseline widths is afforded by fast TOF data acquisition of 0.1 s/spectrum, resulting in a reduction in the baseline width, 1.6 s, of sulfanilamide in a four-compound mixture that is more than 2-fold greater than that achieved at the slower data acquisition of 0.5 s/spectrum. The resulting increase in resolution and improved peak shapes allow automatic integration routines to perform more effectively. For most classes of compounds amenable to high performance liquid chromatography, including druglike species, steroids, and polymers, the union of SFC with TOF-MS provides the maximum density of chemical information per unit time available with any high-speed chromatographic/mass spectrometric method.

Title: Evaluation of a ristocetin bonded

stationary phase for subcritical Abstract: A stationary phase derived from ristocetin was evaluated for chiral separation in subcritical fluid

37

fluid chromatography of enantiomers.

Authors: Lavison G, Thiebaut D

Journal: Chirality 2003 15(7) 630-636

Address: ESPCI, LECA Paris, France.

chromatography. Separation of various enantiomers having different structures and pK(a) values were investigated using carbon dioxide and polar modifiers. The influence of modifiers, additives, temperature, and mobile phase flow rate on separations is presented. It is concluded that this stationary phase can be used for SFC despite its structural similarity with protein-derived stationary phases that can only be used in HPLC. The separation mechanisms could not be elucidated or predicted using these initial experiments. The separations of warfarin and, especially, efavirenz demonstrate the potential of this type of stationary phase for rapid SFC chiral separations.

Title: Studies on the racemization of a

Stereolabile 5-Aryl-thiazolidinedione

Authors: Welch CJ, Kress MH, Beconi M, Mathre DJ

Journal: Chirality 2003 15 143-147

Address: Merck & Co. Department of Process Research, Merck Research Laboratories, Rahway, New Jersey, USA

Abstract: The enantiomers of the stereolabile peroxisome proliferator-activated receptor (PPAR) agonist, 1, were isolated by preparative chiral chromatography and their absolute configuration established using a combination of chromatographic and NMR methods. Enantiomer interconversion was investigated under a variety of conditions, with rapid racemization being observed in most solvents, including all aqueous systems studied, irrespective of pH. Rapid racemization in both dog and human plasma was confirmed by chiral HPLC with MS detection.

Title: Analysis of long-chain polyprenols

using supercritical fluid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Authors: Bamba T, Fukusaki E, Nakazawa Y,

Abstract: The separation of long-chain polyprenols was successfully achieved using supercritical fluid chromatography (SFC). Each 100-mer greater component was separated using tetrahydrofuran as a mobile phase modifier. The molecular mass distributions derived from SFC analyses agreed with the results of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses. The number-average molecular mass calculated by MALDI-TOF-MS data were also in accord with the results of quantitative 1H-NMR analysis of terminal groups. A combination of SFC

Sato H, Ute K, Kitayama T, Kobayashi A


Address: Osaka University, Department of Biotechnology, Graduate School of Engineering, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

and MALDI-TOF-MS analyses is a powerful tool for the elucidation of the complicated structures of natural polyprenols.

Title: Chiral separation of drugs based

on macrocyclic antibiotics using HPLC, supercritical fluid chromatography (SFC) and capillary electrochromatography (CEC) (Article in Czech)

Authors: Dungelova J, Lehotay J, Rojkovicova T,Cizmarik J

Journal: Ceska Slov Farm. 2003 52(3) 119-125

Address: Slovenskej technickej univerzity, Katedra analytickej chemie Fakulty chemickej a potravinarskej technologie Bratislava.

Abstract: Separation of enantiomers by means of high-performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC), and capillary electrochromatography (CEC) is of great importance in pharmaceutical analyses. In recent years, separation of various types of racemates employs most frequently chiral stationary phases based on macrocyclic antibiotics. This class of chiral selectors includes vancomycin, teikoplanin, ristocetin A, teikoplanin without saccharide components, avoparcin, etc. The review paper describes the properties of selected antibiotics, the effect of chromatographic conditions on enantioselectivity (effect of the composition of the mobile phase, effect of pH of the mobile phase, effect of temperature), and the study of possible mechanisms of interaction, which play an important role in the separation of enantiomers. Examples of the use of macrocyclic antibiotics in the separation of various compounds by means of the HPLC, SFC, and CEC methods follow.

Title: Design for on-line coupling of

supercritical fluid extraction with liquid chromatography: quantitative analysis of

Abstract: Supercritical fluid extraction (SFE) was directly coupled with high-performance liquid chromatography (LC) via the simplest interfacesonly one six-port injection valve. By using water to eliminate decompressed CO2 gas in the solid-phase octadecylsilica trap, high extraction recovery

39

polynuclear aromatic hydrocarbons in a solid matrix

Authors: Wang Z, Ashraf-Khorassani M, Taylor LT


Address: Virginia Tech, Department of Chemistry, Blacksburg, Virginia 24061-0212 USA

(g95%) of polynuclear aromatic hydrocarbons (PAHs) from a sand matrix was achieved under optimized conditions. The volume of rinse water had little influence on the recovery, due to the very low solubility of PAHs in water and the sorption properties of the C-18 trap. Different amounts of sand matrix with a fixed mass of analytes have also been tested. No decrease in recovery was found when the matrix (sand) increased from 1 to 10 g. Methanol and acetone were used as a CO2 modifier to enhance the extraction efficiency. Finally, PAHs in naturally contaminated soil were successfully extracted and quantitatively determined by this hyphenated system. Compared to the EPA method (Soxhlet extraction following by GC/MS), on-line SFE-LC gave precise results in a much shorter time.

Title: Chromatography with dynamically

created liquid “stationary” phases: methanol and carbon dioxide

Authors: Luo Z, Xiong Y, Parcher JF

Journal: Anal Chem. 2003 75(3) 622-626

Address: University of Mississippi, Chemistry Department, Mississippi 38677, USA

Abstract: Liquid films composed of binary mixtures of carbon dioxide and methanol were created in empty capillary columns to produce effective stationary phases for chromatography. Under certain conditions of temperature, pressure, and stoichiometric composition, a binary mobile phase composed of CO2 and an organic liquid, such as methanol, can form two immiscible (gas and liquid) phases within a chromatographic column. The two phases can coexist in dynamic equilibrium with the liquid phase migrating through the column at a slower velocity than the gas phase. The liquid phase, composed of methanol saturated with carbon dioxide, acted as a chromatographic stationary phase while the gas phase, composed of carbon dioxide saturated with methanol, acted as a chromatographic mobile phase. The exact conditions necessary for the formation of two phase systems were determined from three-dimensional (P, T, XY) phase diagrams calculated from the Peng-Robinson cubic equation-of-state using one-parameter mixing rules. Separations of simple hydrocarbon mixtures are illustrated under various experimental conditions.

Title: Reaction monitoring of aliphatic

amines in supercritical carbon dioxide by proton nuclear magnetic resonance spectroscopy and implications for supercritical

Abstract: In the recent years, it has repeatedly been stated that amines react with CO2 and can therefore not be chromatographed under supercritical conditions with CO2. The aim of the present work is to elucidate the structural requirements and conditions that can lead to the reaction of an amine analyte with CO2 and, if this occurs, the structure of the formed product. The use of on-line nuclear

fluid chromatography. Authors: Fischer H, Gyllenhaal O, Vessman J,

Albert K Journal: Anal Chem. 2003

75(3) 622-626

Address: Universitat Tubringen, Institut fur Organische Chemie, Auf der Morgenstelle 18, D-72076 Tubingen, Germany.

magnetic resonance (NMR) spectroscopy with a flow probe for supercritical fluid chromatography (SFC) enables the investigation of these unstable analytes in supercritical mediums. Several alkyl-substituted secondary benzylamines and some primary aromatic amines were dissolved in supercritical CO2 and investigated by employing on-line SFC-1H NMR spectroscopy. It was found that the condition of carbamic acid formation depends on the steric properties of the substituents of the amine. A 2-isopropylamino alcohol compound, metoprolol, was also investigated with the setup. No carbamic acid could be detected with the present conditions.

Title: Separation of albendazole

sulfoxide enantiomers by chiral supercritical-fluid chromatography.

Authors: del Nozal MJ, Toribio L, Bernal JL, Nieto EM, Jimenez JJ

Journal: J Biochem Biophys Methods. 2002 54(1-3) 339-345


Abstract: The enantioseparation of albendazole sulfoxide (ABZSO) by chiral supercritical-fluid chromatography (SFC) on two columns, based on the polysaccharide derivatives Chiralpak AD and Chiralcel OD, was studied. The effect of different modifiers, methanol, ethanol, 2-propanol, and acetonitrile, was examined. The results showed that ABZSO can be separated on both columns, using an alcohol-type modifier. Using the Chiralpak AD column, the best results were obtained with 2-propanol and, in the case of the Chiralcel OD, with methanol.

Title: Separation of antifungal chiral

drugs by SFC and HPLC: a comparative study.

Authors: Bernal JL, Toribio L, del Nozal MJ, Nieto EM, Montequi MI

Abstract: The enantiomeric separation of several compounds, including an antifungal drug and several of its precursors, using HPLC and SFC is described in this work. The columns employed were based on polysaccharide derivatives and the results show that most of the separations obtained by SFC are

41



better, in terms of high resolution and short analysis time, than those obtained by HPLC. Only one compound could not be resolved using SFC but, in this case, HPLC provided baseline resolution.

Title: Enantiomeric separations of

amino alcohols by packed-column SFC on Hypercarb with L-(+)-tartaric acid as chiral selector.

Authors: Gyllenhaal O, Karlsson A


Address: AstraZeneca R&D Molndal, Analytical Chemistry, Pharmaceutical R&D, S-431 83 Molndal, Sweden.

Abstract: The use of L-(+)-tartaric acid as a chiral mobile phase additive (CMPA) has been investigated in a packed-column SFC system. The CMPA, carbon dioxide, and methanol, containing a high concentration of aliphatic amine additive, were used as the mobile phase and Hypercarb as support [Gyllenhaal O., Karlsson A., SFC of metoprolol and other amino alcohols on Hypercarb (in preparation)]. Good enantioselectivities were obtained for tertiary amine homologues of 2-amino alcohols, used as beta-adrenoreceptor-blocking drugs. Moderate selectivities were observed for aromatic compounds having a second substituent in the ortho-position. The overall retention was influenced by the aromaticity of the analytes as well as the presence of free electron pairs in the molecule. Increased concentrations of CMPA gave higher retention and also increased the enantioselectivity. The practical utility of this present enantioselective system was demonstrated on one batch of (S)-metoprolol that was N-methylated with methyl iodide. The enantiomeric separation was accomplished within 10 min.

Title: Simulated moving bed

chromatography with supercritical fluids for the resolution of bi-naphthol enantiomers and phytol isomers.

Authors: Johannsen M, Peper S, Depta A

Journal: J Biochem Biophys Methods. 2002

Abstract: The combination of the simulated moving bed (SMB) technique with supercritical fluid chromatography (SFC) leads to a process with unique features. Besides the known advantages of the SMB process, the use of supercritical carbon dioxide as the mobile phase offers the advantages of reduction in organic solvents and an easy eluent/solute separation. Because of the low viscosity and high diffusion coefficients of supercritical fluids, a high efficiency is possible. The steps of process development for SMB SFC are presented using the separations of the bi-naphthol

54(1-3) 85-102

Address: Technische Universitat Hamburg-Harburg, Arbeitsbereich Verfahrenstechnik II, VT II, 6-03, Eissendorfer Strasse 38, Hamburg D-21073, Germany.

enantiomers and phytol isomers as examples. The development of a packed column SFC method at an analytical scale is shown for the separation of the bi-naphthol enantiomers on a chiral stationary phase and CO(2) with a modifier as the mobile phase. The influence of the modifier, modifier content, and column configuration on productivity of the SMB SFC process was investigated by simulation. The first set of experiments was performed in the SMB separation of phytol isomers at low concentration to test the feasibility of the SMB SFC high purity separation of the binary mixtures. In the second set of experiments, the productivity of the process was increased by increasing the feed concentration up to 54 grams feed per liter stationary phase (SP) and hour (g(feed)/l(SP) h).

Title: Super/subcritical fluid

chromatography chiral separations with macrocyclic glycopeptide stationary phases.

Authors: Liu Y, Berthod A, Mitchell CR, Xiao TL, Zhang B, Armstrong DW


Address: Iowa State University, Department of Chemistry, Gilman Hall, Ames, IA 50011, USA.

Abstract: The chiral recognition capabilities of three macrocyclic glycopeptide chiral selectors, namely teicoplanin (Chirobiotic T), its aglycone (Chirobiotic TAG) and ristocetin (Chirobiotic R), were evaluated with supercritical and subcritical fluid mobile phases. A set of 111 chiral compounds including heterocycles, analgesics (nonsteroidal antiinflamatory compounds), beta-blockers, sulfoxides, N-protected amino acids and native amino acids was separated on the three chiral stationary phases (CSPs). All separations were done with an outlet pressure regulated at 100 bar, 31oC and at 4 ml/min. Various amounts of methanol ranging from 7 to 67% (v/v) were added to the carbon dioxide along with small amounts (0.1 to 0.5%, v/v) of triethylamine and/or trifluoroacetic acid. The Chirobiotic TAG CSP was the most effective closely followed by the Chirobiotic T column. Both columns were able to separate, partially or fully, 92% of the enantiomers of the compound set. The ristocetin chiral selector could partially or baseline resolve only 60% of the enantiomers tested. All separations were done in less than 15 min and 70% were done in less than 4 min. The speed of the separations is the main advantage of the use of SFC compared to normal-phase HPLC. In addition, SFC is advantageous for preparative separations with easy solute recovery and solvent disposal.

Title: Characterization of triglycerides in

vegetable oils by silver-ion packed-column supercritical fluid

Abstract: Characterization of triglycerides in vegetable oils was achieved by silver-ion packed-column supercritical fluid chromatography (SI-pSFC) with mass spectrometric detection. Hyphenation was

43

chromatography coupled to mass spectroscopy with atmospheric pressure chemical ionization and coordination ion spray.

Authors: Sandra P, Medvedovici A, Zhao Y, David F


Address: University of Stellenbosch, Department of Chemistry, CENSSUS, Private Bag X1, Matieland 7602, South Africa.

made using commercially available liquid chromatography-mass spectrometry (LC-MS) interfaces without any modification. A make-up fluid was delivered through a T-piece placed before or after the SFC restrictor by means of a high pressure pump. Atmospheric pressure chemical ionization (APCI) and coordination ion spray (CIS) with silver ions were used as ionization modes. Compared to UV detection, the sensitivity was increased by a factor of 100. Both ionization modes are generating similar structural information. Molecular ions [M-H]+ or [M-Ag(-)] are observed in the mass spectra with exception of the saturated triglycerides for which only CIS gives intense molecular ions. The position at which the fatty acids are esterified to the glycerol backbone can be elucidated by pSFC-APCI although it remains speculative whether this is valid for highly unsaturated triglycerides because reference compounds are not available to proof this.

Title: Separation of stereoisomers of

several furan derivatives by capillary gas chromatography–mass spectrometry, supercritical fluid chromatography, and liquid chromatography using chiral stationary phases

Authors: Kasai HF, Tsubuki M, Takahashi K, Shirao M, Matsumoto Y, Honda T, Seyama Y


Address: Hoshi University, Faculty of Pharmaceutical Sciences, Ebara 2-4-41, Shinagawa-ku, Tokyo 142-8501,

Abstract: The direct separation of several stereoisomers (enantiomers and geometrical isomers) of furan derivatives, important intermediates for the synthesis of physiologically active natural products, was achieved using capillary gas chromatography/mass spectrometry with a per-O-methyl-b-cyclodextrin, supercritical fluid chromatography and high-performance liquid chromatography with a tris (3,5-dimethylphenylcarbamate) of cellulose or amylose for the chiral stational phases, respectively. The temperature dependence of the peak resolution (R ) and the retention factor (k) over the range of 110–1308 s was studied using crotyl furfuryl ether in gas chromatography. Successive increases in the R value and of the difference s between the k value of the E-isomer and the k value of the Z-isomer were observed when the gradient temperature was decreased. The per-O-methyl-b-cyclodextrin column was suitable for use with volatile furan ethers whose molecular masses are between 150 and 180. In conclusion, the separation of thermally unstable furan derivatives was accomplished using supercritical fluid chromatography and high-performance liquid chromatography.

Japan

Title: Characterization of low molecular weight alkoxylated polymers using long column SFC/MS and an image analysis based quantitation approach.

Authors: Pinkston JD, Marapane SB, Jordan GT, Clair BD

Journal: J Am Soc Mass Spectrom. 2002 13(10) 1195-1208

Address: The Procter and Gamble Company, Miami Valley Laboratories, Cincinnati, Ohio 45253-8707, USA.

Abstract: The utility of low viscosity mobile phases and long chromatographic columns for complex polymer analysis is demonstrated. We use long column supercritical fluid chromatography/mass spectrometry (SFC/MS) with electrospray ionization (ESI) to characterize a variety of complex, low molecular weight polymers. When quantitative analysis is desired, the resulting three-dimensional (time, intensity, and mass-to-charge ratio [m/z]) data are converted to images. Custom image analysis software is used to detect and integrate peaks in arbitrarily defined regions of the time-m/z map. These integrated peak volumes can be used to quantitate distinct component classes of the polymer mixtures.

Title: Analysis of fatty acids in foods by

supercritical fluid chromatography Authors: Señoráns FJ, Ibañez E

Journal: Analytica Chimica Acta 2002 465 131–144

Address: Instituto de Fermentaciones Industriales, Departamento de Caracterización de Alimentos, C.S.I.C., Juan de la Cierva 3 28006 Madrid, Spain

Abstract: The separation of fatty acids (as fatty acid methyl esters, FAMEs or free fatty acids, FFAs) by supercritical fluid chromatography (SFC) is reviewed. Different analytical approaches are discussed to separate both FAMEs and FFAs, among these approaches, the tuning of the mobile and the stationary phase are reviewed for open tubular, packed and packed capillary SFC. The approach of tuning the polarity of the stationary phase as a way of increasing the range of polar compounds analyzed by SFC using pure CO2 is widely discussed in this review for compounds such as FFAs avoiding the drawbacks associated to the use of modifiers in SFC. Applications of the analysis of FAMEs and FFAs in different foods are also reviewed.

Title: Determination of the Abstract:

The first-order kinetic equation for irreversible reactions was used to determine the enantiomerization

45

enantiomerization energy barrier of some 3-hydroxy-1,4-benzodiazepine drugs by supercritical fluid chromatography.

Authors: Oswald P, Desmet K, Sandra P, Krupcik J, Majek P, Armstrong DW

Journal: J Chromatogr B Analyt Technol Biomed Life Sci. 2002 779(2) 283-295

Address: Slovak University of Technology, Department of Analytical Chemistry, Radlinskeho 9, Bratislava 81237, Slovakia.

barrier of some of 3-hydroxy-1,4-benzodiazepine enantiomers by supercritical fluid chromatography (SFC). The racemates of lorazepam, oxazepam and temazepam were separated by SFC on chiral (R,R)-Whelk-O1 column with supercritical carbon dioxide containing 12.5% methanol and 0.5% diethylamine as a mobile phase. Peak areas of enantiomers prior to (A(A0), A(B0)) and after the separation (A(A), A(B)), used for calculation of the enantiomerization barrier, were determined by computer-assisted peak deconvolution of peak clusters from the chromatograms. It was demonstrated for the first time that using a model for a four-peak cluster produces height precise results, and most closely approximates the published results. The kinetic equation for irreversible reactions was used to determine apparent enantiomerization rate constants. The dependence of the apparent enatiomerization barrier (deltaG(app)(A-->B), deltaG(app)(B-->A)) on temperature was used to determine apparent activation enthalpy (deltaH(app)(R-->S), deltaH(app)(S-->R)) and entropy (deltaS(app)(R-->S), deltaS(app)(S-->R)) for all studied benzodiazepines.


coupled to electrospray mass spectrometry: a powerful tool for the analysis of chiral mixtures

Authors: Hamdan M, Garzotti M

Journal: J. Chromatogr. B 2002 770 53–61

Address: GlaxoSmithKline SpA, Computational, Analytical and Structural Sciences, Medicines Research Centre, Via Fleming 4, 37135, Verona, Italy

Abstract: Supercritical fluid chromatography coupled to a hybrid mass spectrometer (Q-Tof2) equipped with electrospray ion source has been used to separate and characterise a wide range of pharmaceutical racemates. We have chosen diverse molecular structures to demonstrate the potential of such experimental arrangement for high throughput analyses. The use of three different chiral stationary phases and different pressure / temperature working conditions provided clear indications on how such a high throughput method can be developed. The use of mass spectrometry was found to be essential for an unambiguous assignment of the eluting components particularly in the case of complex mixtures. The direct coupling of both systems without the need for a special interface resulted in similar peak shapes and peak widths in the UV and total ion current (TIC) chromatograms.

Title: Determination of valepotriates. Authors: Bos R, Woerdenbag HJ, Pras N


Address: Groningen Research Institute of Pharmacy (GUIDE), Department of Pharmaceutical Biology, The Netherlands.

Abstract: In this paper an overview is given of qualitative and quantitative methods of analysis used for valepotriates. Methods like spectophotometry, titrimetry, TLC, GC, HPLC, MS, CE as well as p-SFC have been applied. Today HPLC is the method of choice. The usefulness of the individual methods are discussed.

Title: Sample deposition device for off-

line combination of supercritical fluid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Authors: Planeta J, Rehulka P, Chmelik J


Address: Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Brno.

Abstract: A new sample deposition device for off-line SFC-MALDI combination of supercritical fluid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was assembled. This device was successfully applied to the detailed characterization of synthetic silicone oils. SFC was used to separate samples of silicone oils on micropacked capillary columns and to determine their molecular mass distribution. The separated fractions for the identification studies were obtained from SFC runs at defined time intervals. Using the constructed deposition device, these fractions were sprayed directly from the restrictor on the target probe covered with a proper matrix. MALDI-TOF MS was used for the identification of individual oligomers in the separated fractions and also in the unfractionated sample. The determined molecular mass distributions based on supercritical fluid chromatography with flame ionization detector, MALDI-TOF MS, and combined SFC-MALDI measurements were compared and the results were in a good agreement. The sample deposition device is based on a common plotter unit, complemented by a microcontroller PIC16C84. The unit is connected by an RS-232 interface to a PC with the main control software running under MS Windows. The new sample deposition device made the off-line combination SFC-MALDI simpler, faster, and more sensitive.

Title: Supercritical fluid and unified

chromatography Authors: Chester TL, Pinkston JD

Abstract:

47


Address: The Procter & Gamble Company, Miami Valley Laboratories, P.O. Box 538707, Cincinnati, Ohio 45253-8707

Title: Direct injection of aqueous

samples in packed column supercritical fluid chromatography of isosorbide-5-mononitrate from drug release testing.

Authors: Gyllenhaal O, Hulthe J

Journal: J Pharm Biomed Anal. 2002 29(1-2) 381-386

Address: AstraZeneca R&D Molndal, Analytical and Technical Development, S-431 83 Molndal, Sweden.

Abstract: A method for the analysis of aqueous samples of isosorbide-5-mononitrate (5-ISMN) is presented. It is based on packed column supercritical fluid chromatography (SFC) using 20% of 2-propanol in carbon dioxide as the mobile phase and a diol silica column as the stationary phase. Using the described conditions it is possible to quantitate 5-ISMN released from Imdur tablets in gastric media. The precision upon repeated injections was 2% (RSD) at the 20 microg/ml level (n=8), using peak height measurements, when the solution was circulated through the sample loop of the injector. Samples from drug release testing that had been analyzed with reversed phase LC were analyzed with the present method and the results agreed well. It is also possible to monitor the drug released in a dissolution-testing vessel through direct on-line continuous loading (recirculation) of the sample loop of the SFC instrument.

Title: Characterization of oligomeric

polypropyleneglycol acrylate by GC, SFC and MALDI-TOF-MS.

Authors: Matsunaga M, Matsushima Y, Yokoi H, Ohtani H, Tsuge S

Journal: Anal Sci. 2002 18(3) 277-281

Address: Toagosei Co, Ltd, Nagoya Reseach & Development

Abstract: Polypropyleneglycol acrylate (PGA), one of the typical acrylic oligomers manufactured industrially, was comprehensively characterized by gas chromatography (GC), supercritical fluid chromatography (SFC) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The homologous series of polypropyleneglycol diacrylate (DA), polypropyleneglycol monoacrylate (MA), and unreacted polypropyleneglycol (PG) were observed as Na adducts in the MALDI-MS spectra of the PGA samples. The relative intensities of these peaks reflect the distributions of the homologues, although their accurate quantification was generally difficult because of change in the ionization efficiency depending on the chemical structure and the

Institute, Minato, Japan

molecular weight of the species. On the other hand, the DA and the MA homologues were observed in the chromatograms obtained by SFC in a temperature-programming mode, while the PG homologues were not detected under the given SFC conditions using UV detection. Here, the determination of the degree of polymerization of each component in the chromatograms was accomplished through SFC fractionation for the corresponding peaks, followed again by MALDI-TOF-MS measurement. Furthermore, most of the components in the PGA samples were almost completely separated in the resulting gas chromatograms, and their unequivocal assignments were made also using the retention data on the gas chromatograms of the SFC fractions. As for the quantitative analysis, the relative abundances among DA, MA and PG for lower degrees of polymerization can be easily calculated based on the observed gas chromatograms, whereas the distribution of DA and MA can be estimated from the observed SFC data even for the relatively higher molecular weight fractions, which are generally difficult to determine accurately by GC because of their lower volatility. These results demonstrated that even the complex PGA samples were able to be characterized in detail by using GC, SFC and MALDI-TOF-MS complementarily.

Title: High-throughput techniques for

compound characterization and purification.

Authors: Kyranos JN, Cai H, Zhang B, Goetzinger WK

Journal: Curr Opin Drug Discov Devel. 2001 4(6) 719-28

Address: ArQule Inc 19 Presidential Way Wobum, MA 01801, USA

Abstract: A new paradigm in drug discovery is the synthesis of structurally diverse collections of compounds, so-called libraries, followed by high-throughput biological screening. High-throughput characterization and purification techniques are required to provide high-quality compounds and reliable biological data, which has led to the development of faster methods, system automation and parallel approaches. This review summarizes recent advances in support of analytical characterization and preparative purification technologies. Notably, mass spectrometry (MS) and supercritical fluid chromatography (SFC) are among the areas where new developments have had a major impact on defining these high-throughput applications.

Title: A study of polyethoxylated

alkylphenols by packed column supercritical fluid

Abstract: Alkylphenol polyethoxylates (APEs) are a widely used group of nonionic surfactants in commercial production. Characterization of the composition of APE mixtures can be exploited for the

49

chromatography. Authors: Hoffman BJ, Taylor LT

Journal: J Chromatogr Sci. 2002 40(2) 61-68

Address: Virginia Tech, Department of Chemistry, Blacksburg 24061-0212, USA.

determination of their most effective uses. In this study sample mixtures contain nonylphenol polyethoxylates and octylphenol polyethoxylates. The separation of individual alkylphenols by ethoxylate units is performed by supercritical fluid chromatography (SFC)-UV as well as normal-phase high-performance liquid chromatographic (HPLC)-UV employing packed columns. The stationary phase and column length are varied in the SFC setup to produce the most favorable separation conditions. Additionally, combinations of packed columns of different stationary phases are tested. The combination of a diol and a cyano column is found to produce optimal results. An advantage of using packed columns instead of capillary columns is the ability to inject large amounts of sample and thus collect eluted fractions. In this regard, fractions from SFC runs are collected and analyzed by flow injection analysis-electrospray ionization-mass spectroscopy in order to positively identify the composition of the fractions. In comparing the separation of APE mixtures by SFC and HPLC, it is found that SFC provides shorter retention times with similar resolution. In addition, less solvent waste is produced using SFC.

Title: The SFC comeback.

Pharmaceuticals give supercritical fluid chromatography a fighting chance.

Authors: Harris CM

Journal: Anal Chem. 2002 74(3) 87A-91A

Address:

Abstract: Review

Title: Chiral separation by

chromatographic and electromigration techniques. A review.

Authors: Gubitz G, Schmid MG

Abstract: This review gives a survey of different chiral separation principles and their use in high-performance liquid chromatography (HPLC), gas chromatography (GC), supercritical fluid chromatography (SFC), thin-layer chromatography (TLC), capillary electrophoresis (CE) and capillary electrochromatography (CEC) highlighting new developments and innovative techniques. The

Journal: Biopharm Drug Dispos. 2001 22(7-8) 291-336

Address: Institute of Pharmaceutical Chemistry and Pharmaceutical Technology, Karl-Franzens University, Universitatsplatz 1, A-8010 Graz, Austria

mechanisms of the different separation principles are briefly discussed and some selected applications are shown.

Title: Supercritical fluid extraction and

chromatography for fat-soluble vitamin analysis

Authors: Turnera C, Kingb JW, Mathiassona L


Address: Lund University, Department of Analytical Chemistry, P.O. Box 124, S-221 00 Lund, Sweden

Abstract: Extraction and chromatographic separation of fat-soluble vitamins is a challenging task, due to the sensitivity of these compounds towards light, oxygen, heat and pH. In light of this, supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC) are attractive techniques as they function at considerably milder conditions than conventional solvent-based analytical techniques. Moreover, supercritical techniques consume much less amounts of organic solvents than conventional ones. This review gives a brief description of suitable supercritical media as well as basic theory on SFE and SFC processes. Furthermore, guidelines are provided for optimizing the important extraction and separation parameters to facilitate a successful method development. Finally, applications employing SFE and/or SFC for fat-soluble vitamin enrichment and final determination are reviewed.

Title: Separation of enantiomers of

ibuprofen on chiral stationary phases by packed column supercritical fluid chromatography.

Authors: Johannsen M


Abstract: A packed column supercritical fluid chromatography (SFC) method for the separation of ibuprofen enantiomers on a chiral stationary phase and CO2 with modifier as mobile phase has been developed at an analytical scale. Among 11 different stationary phases the Kromasil CHI-TBB phase showed by far the best separation properties. The influence of different modifiers, injection solvents, temperature, and pressure, and density of the fluid, respectively, on the separation behavior has been studied. It was found that the separation behavior strongly depends on the type of modifier and the modifier content. Temperature and pressure are of less influence.

51

Address: Technische Universitat Hamburg-Harburg, Arbeitsbereich Verfahrenstechnik II, Germany.

Title: Supercritical fluid extraction and

chromatography for fat-soluble vitamin analysis.

Authors: Turne C, King JW, Mathiasson L


Address: Lund University, Department of Analytical Chemistry, P.O. Box 124, S-221 00 Lund, Sweden.

Abstract: Extraction and chromatographic separation of fat-soluble vitamins is a challenging task, due to the sensitivity of these compounds towards light, oxygen, heat and pH. In light of this, supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC) are attractive techniques as they function at considerably milder conditions than conventional solvent-based analytical techniques. Moreover, supercritical techniques consume much less amounts of organic solvents than conventional ones. This review gives a brief description of suitable supercritical media as well as basic theory on SFE and SFC processes. Furthermore, guidelines are provided for optimizing the important extraction and separation parameters to facilitate a successful method development. Finally, applications employing SFE and/or SFC for fat-soluble vitamin enrichment and final determination are reviewed.

Title: Analysis of non-saponifiable lipids

by super-/subcritical-fluid chromatography.

Authors: Lesellier E


Address: Groupe de chimie analytique de l'Universite Paris Sud (XI), L.E.T.I.A.M.. IUT ORSAY, Plateau de Moulon, 91400 Orsay, France.

Abstract: Because of the particular properties of carbon dioxide or carbon dioxide/modifier mobile phases, super- or subcritical-fluid chromatography (SFC) can be an alternative to more classical chromatographic methods such as gas chromatography (GC) or high-performance liquid chromatography (HPLC) for the separation of unsaponifiable lipids. These fluids can also be helpful in the extraction and/or the concentration steps of sterols, tocopherols or carotenoids from complex samples. Supercritical extraction, off-line prefractionation or semi-preparative supercritical fluid chromatography, carried out before the analysis are described. The effects on separation of analytical parameters such as pressure, nature of and modifier percentage or stationary phase nature are also reported. The performance of capillary, packed or capillary packed columns is discussed, as well as the consequences of their use (choice of stationary phases, type of coupled detector). Numerous examples of fine separations are reported.

Title: Chiral supercritical fluid chromatography on porous graphitic carbon using commercial dimethyl b-cyclodextrins as mobile phase additive

Authors: Salvadora A, Herbreteau B, Dreux M, Karlssonb A, Gyllenhaalb O

Journal: J. Chromatogr. A 2001 929 101-112

Address: Institut de Chimie Organique et Analytique, UPRES A CNRS 6005, Universite d’Orleans, F-45067 Orleans Cedex 02, France

Abstract: Using dimethylated-b-cyclodextrin mixtures (MeCD) as chiral selectors in CO -polar modifier mobile phase and porous 2 graphitic carbon as solid-phase, chiral supercritical (or subcritical) fluid chromatography was performed. The adsorbed quantity of MeCD onto the porous graphitic carbon (Hypercarb) was measured for various chiral selector concentrations using the breakthrough method with evaporative light scattering detector. The effects of MeCD concentration in the mobile phase, the nature of the polar modifier, the outlet pressure, the column temperature and the nature of the commercial MeCD mixture on the retention and the enantioselectivities were studied. For a given solute, the enantioselectivity is greatly dependent on the commercial MeCD mixture used. The retention mechanism was also studied. From the data, we find that the dominant mechanism for the chiral discrimination is the diastereoisomeric complexation in the mobile phase.

Title: Applications of the Chiralpak AD

and Chiralcel OD chiral columns in the enantiomeric separation of several dioxolane compounds by q supercritical fluid chromatography

Authors: Toribio L, del Nozal MJ, Bernal JL, Jimenez JJ, Nieto EM


Address: University of Valladolid, Department of Analytical Chemistry, Faculty of Sciences, Prado de la Magdalena s /n, E-47005 Valladolid, Spain

Abstract: Two chiral columns based on polysaccharide derivatives (Chiralpak AD and Chiralcel OD) have been tested for the chiral separation of several dioxolane compounds, using supercritical fluid chromatography. The compounds studied included ketoconazole and some of its precursors. The effect of the different modifiers and the pressure, on the chromatographic parameters was also evaluated. In general, the alcohol modifiers provided better results than acetonitrile, and all the compounds could be separated with these two columns, but the selection of the column depends on the kind of compound.

53

Title: Mass-directed fractionation and

isolation of pharmaceutical compounds by packed-column supercritical fluid chromatography/mass spectrometry.

Authors: Wang T, Barber M, Hardt I, Kassel DB

Journal: Rapid Commun Mass Spectrom. 2001 15(22) 2067-2075

Address: Dupont Pharmaceutical Research Laboratories, Executive Drive, San Diego, CA 92121, USA

Abstract: An automated packed-column semi-preparative supercritical fluid chromatography/mass spectrometry (SFC/MS) system incorporating mass-directed fraction collection has been designed and implemented as an alternative to preparative HPLC and preparative HPLC/MS (PrepLC/MS) for the purification of pharmaceutical compounds. The system incorporates a single quadrupole mass spectrometer and a supercritical fluid chromatograph. Separations were achieved using a binary solvent system consisting of carbon dioxide and methanol. Purification of SFC-separated compounds was achieved incorporating mass-directed fraction collection, enabling selective isolation of the target molecular weight compound and eliminating the collection of undesired compounds (e.g., by-products, excess starting materials, etc.). Cross contamination between fractions and recoveries of the system were investigated. Mass spectrometer ionization with basic mobile additives is discussed, and examples of preparative SFC/MS chiral separations are presented. Early experiences suggest SFC will be a powerful and complementary technique to HPLC for the purification of pharmaceutical compounds.

Title: Comparison of drug substance

impurity profiles generated with extended length columns during packed-column SFC.

Authors: Roston DA, Ahmed S, Williams D, Catalano T

Journal: J Pharm Biomed Anal. 2001 26(3) 339-355

Address: Pharmacia Research and Development, 4901 Searle Parkway, Skokie, IL 60077, USA.

Abstract: The current study assesses the effect of extending column length during gradient packed column sub/supercritical fluid chromatography (PCSFC) experiments on the detection of known and unknown impurities in a drug substance sample. Quantitative drug substance impurity profiles were generated and compared using multiple column PCSFC and HPLC conditions. Also, chromatographic figures of merit were estimated and compared for components of a standard mixture during PCSFC experiments, which used one column, four columns, and six columns in series.

Title: Determination of molecular mass Abstract:

distribution of silicone oils by supercritical fluid chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and their off-line combination.

Authors: Chmelik J, Planeta J, Rehulka P, Chmelik J

Journal: J Mass Spectrom. 2001 36(7) 760-770

Address: Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Veveri 97, 611 42 Brno, Czech Republic.

Silicone oil samples were characterized by supercritical fluid chromatography (SFC), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI--TOF MS), and their off-line combination. SFC was used to separate samples of silicone oils on micropacked capillary columns. The fractions for the identification studies were obtained from SFC runs at defined time intervals, when the restrictor was pulled out from the chromatographic flame ionization detector (FID) and inserted into a glass vial with acetone. MALDI--TOF MS was used for the identification of individual oligomers in the fractions separated. The molecular mass distributions determined based on SFC and MALDI--TOF MS measurements were compared. From this comparison, it follows that the results are in good agreement. However, certain differences were observed: MALDI--TOF MS was capable of detecting somewhat larger oligomers than the SFC-FID, but the lower molecular mass oligomers were not present in the MALDI spectra. Differences in the region of lower molecular masses can be explained by evaporation of the more volatile low molecular mass oligomers resulting from heating of the sample during the MALDI--TOF MS measurements as a result of the absorption of the laser shot energy. The fact that no high mass discrimination effects of the MALDI--TOF MS measurements, compared with SFC, were observed is very promising for further applications of MALDI--TOF MS in characterizing synthetic polymers of moderate polydispersity.

Title: Determination of

enantiomerization barriers by dynamic and stopped-flow chromatographic methods.

Authors: Trapp O, Schoetz G, Schurig V

Journal: Chirality. 2001 13(8) 403-414

Address: University of Tubingen, Institute of Organic Chemistry, Auf der Morgenstelle 18, 72076 Tubingen, Germany.

Abstract: In recent years, dynamic chromatography and stopped-flow chromatographic techniques have become versatile tools for the determination of enantiomerization and isomerization barriers. Increasing demands for the stereochemical safety of chiral drugs contributed to the rapid development of new techniques. New computer-aided evaluation systems allow the on-line determination of interconversion barriers from the experimental chromatograms. Both dynamic chromatography and stopped-flow chromatography have been applied to the entire range of chromatographic methods (GC, SFC, HPLC, CE).

55

Title: Coupling of a supercritical fluid chromatography system to a hybrid (Q-TOF 2) mass spectrometer: on-line accurate mass measurements.

Authors: Garzotti M, Rovatti L, Hamdan M

Journal: Rapid Commun Mass Spectrom. 2001 15(14) 1187-1190

Address: GlaxoSmithKline Group, Glaxo Wellcome SpA, Medicines Research Centre, Via Fleming 4, 37135 Verona, Italy.

Abstract: This note describes a simple and economical method to couple a supercritical fluid chromatography (SFC) system (Berger Instruments, US) with a high-resolution hybrid mass spectrometer (Q-TOF 2; Micromass, UK). This experimental arrangement has three distinct advantages: (1) coupling between the two systems can be effected without the need for an interface or hardware modifications of either system, (ii) this experimental arrangement provides on-line accurate mass SFC/MS measurements which are indispensable for the characterisation of new chemical entities and unknown metabolites, and (iii) the characteristically fast spectral acquisition rate of the time-of-flight (TOF) analyser renders the present arrangement an important contribution to future semipreparative fraction collection setups which use mass spectrometry as a detector.

Title: Determination of sulfadoxine in

human blood plasma using packed-column supercritical fluid chromatography

Authors: Bhoir SI, Bhoir IC, Bhagwat AM, Sundaresan M

Journal: J Chromatogr B Biomed Sci Appl. 2001 757(1) 39-47

Address: C.B. Patel Research Centre for Chemistry and Biological Sciences, Vile Parle (West), Mumbai, India.

Abstract: A sensitive, rapid, selective and reproducible method has been developed to measure plasma levels of sulfadoxine, 4-Amino-N-(5, 6-dimethoxy-4-pyrimidinyl) benzensulfonamide; in healthy, human volunteers using packed-column super-critical fluid chromatography. Omeprazole, 5-methoxy-2-[[(4-methoxy-3, 5-di-methyl-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazole; was used as the internal standard (i.s.) at 15.0 microg/ml. The drug and the i.s. were extracted from plasma using dichloromethane. Separation of sulfadoxine and i.s. was done on a Nucleosil (250x4.6 mm) 10 microm, RP-C18 column with 7.4% (v/v) methanol-modified supercritical fluid carbon dioxide (2.5 ml/min) as the mobile phase. The column temperature was 40oC and the outlet pressure was set at 8.83 MPa. The detection was done using a UV-Vis detector set at 265 nm. The limit of quantification was 0.50 microg/ml using 1 ml plasma specimen. The mean extraction recovery of the drug from plasma was found to be 94.9%. The SFC method was directly compared to a published HPLC/UV method. With respect to speed and use of organic solvents SFC was found to be superior; while in all other aspects the results were similar to the published technique. The method has been successfully used to estimate the sulfadoxine levels in healthy human volunteers from 0 to 240 h

following an oral dose of 500 mg of sulfadoxine in combination with 25 mg of pyrimethamine.

Title: Increased expression of interleukin-8 and aminopeptidase N by cell-cell contact: interleukin-8 is resistant to degradation by aminopeptidase N/CD13.

Authors: Kehlen A, Egbert I, Thiele K, Fischer K, Riemann D, Langner J

Journal: Eur Cytokine Netw. 2001 12(2) 316-324

Address: Martin Luther University, Institute of Medical Immunology, Magdeburger Strasse 2, D-06097 Halle, Germany.

Abstract: In rheumatic joints, high concentrations of interleukin-8 (IL-8) have been measured in synovial fluid and in pannus tissue. In both locations aminopeptidase N (APN)/CD13, an exopeptidase with reported activity towards IL-8 is also present. The surprising stability of IL-8 in the presence of an alleged L-8-degrading peptidase prompted us to undertake the present study. Cocultivation of fibroblast-like synoviocytes (SFC) with T cells or with T lymphocytic cell membranes, or of T cells with SFC cell membranes, all resulted in increased IL-8 mRNA expression and IL-8 secretion into the medium, and an increase of APN expression on lymphocytes. IL-8 degradation was monitored by Western blots and HPLC. IL-8(72), as a partially processed form, was used throughout this study since it is abundant in tissues and has increased biological activity in comparison to IL-8(77). Thus its degradation/inactivation is considered of high biological significance. Whereas trypsin as a positive control rapidly degraded IL-8, we did not see any IL-8 degradation, either by a variety of soluble APNs, by leucine aminopeptidase or by APN expressed on the surface of SFC, or on ECV304 cells transfected with an APN expression vector. The much more sensitive HPLC technique resulted in negative results as well.

Title: Enantioseparation of

aminoglutethimide and thalidomide by high performance liquid chromatography or supercritical fluid chromatography on mono-2 and mono-6-O-pentenyl-beta-cyclodextrin-based chiral stationary phases.

Authors: Duval R, Leveque H, Prigent Y, Aboul-Enein HY

Journal: Biomed Chromatogr. 2001

Abstract: Mono-2 and mono-6-O-pentenyl-beta-cyclodextrin (mono-2-pent-beta-CD and mono-6-pent-beta-CD), covalently linked to mercaptopropylsilica gel (thiol-Si) through thioether or sulfone linkage, reveal differentiated enantioselectivities in the separation of piperidine-2,6-dione-related drugs, namely aminoglutethimide and thalidomide, in supercritical fluid conditions. Supercritical fluid chromatographic resolution on completely defined mono-cyclodextrin derivative-based chiral stationary phases (CSP) is a method of choice for the separation of aminoglutethimide but not effective for thalidomide. For both high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) conditions, the impact of the position, imposed to be 2 or 6 in our synthetic pathway, of the pentenyl moiety on one of the glucopyranosidics of the CD cage is of crucial importance in the chiral discrimination phenomenon. Additionally, the nature of the

57

15(3) 202-206

Address: ChiralSep S.A., Parc d'Activites de la Boissiere, 76170 La Frenaye, France.

heteroatom present in the spacer arm between the CD and the silica gel, in this case thioether or sulfone functionality, is also essential for the chiral recognition mechanism(s) for the solute enantiomer.

Title: Separation of enantiomers by

open capillary electrochromatography on polysiloxane-bonded permethyl-beta-cyclodextrin.

Authors: Schurig V, Mayer S

Journal: J Biochem Biophys Methods. 2001 48(2) 117-141

Address: Institut fur Organische Chemie, Universitat Tubingen, Auf der Morgenstelle 18, D-72076, Tubingen, Germany.

Abstract: The separation of enantiomers by open capillary electrochromatography (o-CEC) using Chirasil-Dex as chiral stationary phase (CSP) is reviewed. In Chirasil-Dex, permethylated beta-cyclodextrin is linked via a single octamethylene spacer to polydimethylsiloxane. The CSP is coated and thermally immobilized onto the internal surface of a fused-silica column (i.d. 50 microm). Employing a single open-tubular column coated with Chirasil-Dex, a unified enantioselective approach can be realized using the four common chromatographic techniques: o-GC, o-SFC, o-LC and o-CEC. The chiral stationary phase Chirasil-Dex can be combined with a charged cyclodextrin derivative, which is added into the mobile phase. In the resulting dual chiral recognition system, enhancement of enantioselectivity (matched case) or compensation of enantioselectivity (mismatched case) are observed. The overall enantioselectivity is dependent on the sense of enantioselectivity of the selectors chosen and their influence on the electrophoretic and electroosmotic migration of the enantiomers of a selectand. The feasibility to couple chiral o-CEC and ESI/MS is demonstrated for trace analysis of enantiomeric drugs in body fluids.

Title: High-resolution analysis of

polyprenols by supercritical fluid chromatography.

Authors: Bamba T, Fukasaki W, Kajiyama S, Ute K, Kitayama T, Kobayashi A


Address: Osaka University,

Abstract: A high-resolution analysis of polyprenol mixtures was achieved by supercritical fluid chromatography (SFC). The separation of polyprenols was examined on an octadecylsilane-packed column with liquid carbon dioxide as the mobile phase and ethanol as modifier. Using this chromatography system, the resolution of separation (Rs) between octadecaprenol (prenol 18) and nonadecaprenol (prenol 19) was two times higher than that using conventional reversed-phase high-performance liquid chromatography. Our SFC technique allows the advantage of baseline separation of polyprenol samples containing hydrophobic components such as terpenes or fatty acids that are unfavorable for good separation. This method is very useful for the analysis of

Department of Biotechnology, Graduate School of Engineering, Suita, Japan.

structurally close polyprenol analogues of rubber plant metabolites.

Title: On-line packed column

supercritical fluid chromatography - microwave-induced plasma atomic emission.

Authors: Bertoncini F, Thiebaut D, Caude M, Gagean M, Carraze B, Beurdouche P, Duteurtre X


Address: Ecole Superieure de Physique et de Chimie Industrielles de Paris, Laboratoire Environnement et Chimie Analytique, France.

Abstract: Interfacing and evaluation of packed column supercritical fluid chromatography (SFC)-microwave-induced plasma atomic emission detection (AED) is described. Via a flow splitter and an integral restrictor, efficient transfer of solutes from column to detector without band broadening is obtained. Variation of CO2 flow-rate during pressure gradients has little influence on both AED signal and baseline drift while it provides similar sensitivity as in capillary SFC. Continuous introduction of CO2 in the plasma reduces the available range of emission domains; nevertheless the region of detection which is free of CO2 interferences allows selective detection of C1 and Br as reported in this paper.

Title: Enantioseparations in super- and

subcritical fluid chromatography. Authors: Terfloth G


Address: SmithKline Beecham Pharmaceuticals, Research and Development, King of Prussia, PA 19406, USA.

Abstract: The separation of chiral compounds by sub- and supercritical fluid chromatography has been a field of great progress since the first demonstration of a chiral separation by SFC in 1985. Almost all of the chiral selectors used in gas or liquid chromatography have been successfully applied to sub-/supercritical chromatography. Easier and faster method development, high efficiency, superior and rapid separations of a wide variety of analytes, extended-temperature capability, analytical and preparative-scale equipment improvements and a selection of detection options have been reported.

59

Title: New device for controlling the amount of methanol in carbon dioxide mobile phase for supercritical fluid chromatography.

Authors: Pyo D

Journal: Microchemical Journal 2001 68 183-188

Address: Kangwon National University, Department of Chemistry, 200-701, Chuncheon, South Korea.

Abstract: A new device to accurately deliver a small amount of methanol into supercritical carbon dioxide fluid is described. Carbon dioxide, the most widely used mobile phase in supercritical fluid chromatography, is a relatively non-polar fluid, and hence the addition of a small amount of methanol could change the solvent strength of the mobile phase. In this work, supercritical CO2 and methanol are delivered from the pump to a 100- l mixing chamber in which a small magnetic bar is rotating. After passing through the mixing chamber, supercritical CO2 is changed to a new mobile phase with different polarity. The modified mobile phase was successfully used for the separations of polar compounds and polyaromatic hydrocarbons (PAHs).

Title: Application of molecularly

imprinted polymers in supercritical fluid chromatography.

Authors: Ellwanger A, Owens PK, Karlsson L, Bayoudh S, Cormack P, Sherrington D, Sellergren B


Address: AstraZeneca R&D Product Analysis I, Analytical Development, Molndal, Sweden.

Abstract: Molecularly imprinted polymers (MIPs), for the templates free base racemic propranolol and the L-enantiomer of phenylalanine anilide (L-PA), were investigated as stationary phases in supercritical fluid chromatography (SFC). Large retention differences were observed on the propranolol MIP for both the template molecule and the structural analogue metoprolol compared to that observed on the corresponding blank polymer. Mobile phase composition and solute concentration were found to affect this retention behaviour. The phenylalanine anilide MIP (L-PA MIP) was found to be enantioselective in SFC with stronger retention observed for the template enantiomer. Throughout the study, characteristic imprinting peak shapes for the stronger retained template molecule were observed for both MIPs examined. After a number of days under supercritical fluid conditions, the performance of the photochemically initiated L-PA MIP was found to significantly deteriorate whereas the thermally initiated propranolol MIP revealed only small changes in its separation performance after a long term of operation. The separation behaviour of these two MIPs in SFC was compared with results obtained on the same columns in high-performance liquid chromatography (HPLC) both before and after their application in SFC.

Title: Chiral separation of four 1,3- Abstract:

dioxolane derivatives by supercritical fluid chromatography on an amylose-based column

Authors: Toribio L, del Nozal MJ, Bernal JL, Jimenez JJ, Serna ML


Address: University of Valladolid, Department of Analytical Chemistry, Faculty of Sciences, Spain

The chiral separation of four 1,3-dioxolane derivatives by supercritical fluid chromatography on an amylose-based column is described. The effects of mobile phase composition, temperature and pressure have been investigated. The nature of the modifier is the parameter which has the highest impact on the chiral resolution and it is more important than the polarity of the mobile phase. The organic modifier used for the best enantiomeric separation was different for each compound, because it depends strongly on the molecular structure of the compound.

Title: Expression and distribution of

GABAA receptor subtypes in human alcoholic cerebral cortex.

Authors: Buckley ST, Eckert AL, Dodd PR

Journal: Ann N Y Acad Sci. 2000 914 58-64

Address: University of Queensland, Department of Biochemistry, Brisbane, Australia.

Abstract: Long-term alcohol abuse is known to target specific areas of the brain such as the superior frontal cortex (SFC), resulting in neuronal cell loss. Abnormal transmission of the inhibitory neurotransmitter GABA may contribute to this damage. Previous work in our laboratory has found differential expression and distribution of certain a subunit genes of the GABAA receptor in the SFC of human alcoholic brain, suggesting that differences in GABAA receptor subunit expression could give rise to the locally altered GABAA pharmacology which is associated with alcohol abuse. A competitive RT-PCR assay has been developed to study the expression of the GABAA receptor beta-subunit genes beta1, beta2, and beta3. A single set of primers homologous to all three beta isoform sequences has been shown to amplify each of the beta isoforms from mRNA isolated from human brain tissue obtained at autopsy. An internal standard has been designed which is identical to the target except for a 61-bp deletion and a unique restriction enzyme (RE) site. This is co-amplified with the target sequences to allow amplification efficiency to be assessed and thus enable the quantitation of gene expression. A range of GABAA receptor ligands were used to look at differential distribution of receptor subtypes in the cortical laminae by autoradiography. Differences in distribution of the ligands were demonstrated, consistent with a hypothesis of alcohol-induced variations in the expression of receptor subunits.

61

Title: Modifier effects on column

efficiency in packed-column supercritical fluid chromatography

Authors: Zou W, Dorsey JG, Chester TL


Address: Florida State University, Department of Chemistry, Tallahassee 32306-4390, USA.

Abstract: We investigate the effects on column efficiency of methanol, acetonitrile, ethanol, and 1-propanol used as modifiers in packed-column SFC. C-18, phenyl, and cyano columns were used with both nonpolar and polar solutes. For highly retained nonpolar solutes, addition of modifier significantly increased apparent column efficiency, especially for the C-18 column. For polar solutes, the presence of modifier dramatically improved retention and efficiency with an apparent efficiency dependence on modifier type and amount. Temperature and pressure effects on efficiency were also studied.

Title: Enantioselective supercritical fluid

chromatography using ristocetin A chiral stationary phases.

Authors: Svensson LA, Owens PK

Journal: Analyst. 2000 125(6) 1037-1039

Address: AstraZeneca R&D Analytical Development, Molndal, Sweden.

Abstract: Racemic mixtures of five acidic drugs have been successfully separated by supercritical fluid chromatography (SFC) using macrocyclic antibiotic chiral stationary phases (CSPs). A ristocetin A CSP has been prepared 'in-house' and effectively applied in packed capillary SFC to separate the enantiomers of dichlorprop (R(s) = 1.4), ketoprofen (R(s) = 0.9) and warfarin (R(s) = 0.9). The commercial ristocetin A CSP (Chirobiotic R) was subsequently studied in packed column SFC with similar results where the enantiomers of warfarin (R(s) = 2.2), coumachlor (R(s) = 2.5) and thalidomide (R(s) = 0.6) were separated. Interestingly, differences were observed between the two differently immobilised CSPs where the enantiomers of dichlorprop and ketoprofen, which were separated on the 'in-house' CSP, could not be separated on the commercial phase.

Title: Determination of ascorbigens in

autolysates of various Brassica species using supercritical fluid chromatography

Authors: Buskov S, Hansen LB, Olsen CE, Sorensen JC, Sorensen H, Sorensen S

Journal: J Agric Food Chem. 2000

Abstract: A new method of analysis based on normal phase supercritical fluid chromatography (SFC) has been developed for investigation of ascorbigens [2-C-(indol-3-ylmethyl)-beta-L-xylo-3-hexulofuranosonic acid gamma-lactone derivatives]. This method has been adapted to preparative isolation and quantitative determinations of individual ascorbigens comprising ascorbigen, neoascorbigen, and 4-methoxyascorbigen. The structures of these compounds have been revealed from 1D ((1)H, (13)C) and 2D (COSY, HMQC, HMBC) NMR experiments. The developed SFC

48(7) 2693-2701

Address: Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.

method had an acceptable linearity for the ascorbigens with correlation coefficients (R(2)) > 0.9995 (n = 10) in the range of 0.13-4.9 nmol injected, detection limits were below 13 pmol, retention time stabilities were excellent, and relative response factors have been determined. The SFC method has been used for determination of ascorbigens produced during autolysis of indol-3-ylmethylglucosinolates in various Brassica vegetables and rapeseed seedlings. Generally, 30-60% of the indol-3-ylmethylglucosinolates in the plants were transformed into ascorbigens, with the concentration in autolysates varying from 0.51 +/- 0.002 to 3.72 +/- 0.21 micromol/g of dry weight (DW) for ascorbigen, from 0.05 +/- 0.01 to 2.42 +/- 0.23 micromol/g of DW for neoascorbigen, and from 0.03 +/- 0.002 to 0.84 +/- 0.07 micromol/g of DW for 4-methoxyascorbigen.

Title: Polar organic phase liquid

chromatography with packed capillary columns using a vancomycin chiral stationary phase

Authors: Svensson LA, Donnecke J, Karlsson KE, Karlsson A, Vessman J


Address: Biomedical Center, Department of Analytical Pharmaceutical Chemistry, Uppsala, Sweden.

Abstract: Vancomycin immobilized on silica served as the chiral stationary phase (CSP) in this investigation with polar organic solvents as the mobile phase in liquid chromatography (LC). It was shown that trace amounts of water were beneficial for improving peak shape and efficiency. To regulate the retention and selectivity an acid and/or base were added to the mobile phase where an excess of acid was shown to be preferential for enantioseparation. An unusual increase in selectivity with increasing temperature was shown for the acidic drug, thalidomide. Additionally, nonlinear van't Hoff plots were obtained for metoprolol enantiomers that showed increased retention with increasing temperature. Metoprolol also showed unusual behavior in the polar organic phase when water was added to resemble reversed-phase chromatography, with minimum retention observed at high water or high methanol concentrations. In both instances a high degree of electrostatic interaction between metoprolol and vancomycin was concluded. Metoprolol and ten of its analogs were examined on this CSP to evaluate the enantiorecognition process. A comparison in enantioselectivity for a number of acidic and basic drugs using this CSP was also carried out using the polar organic phase, reversed phase, and normal phase LC which were all compared to the results obtained in supercritical fluid chromatography (SFC). Polar organic phase LC offered a better separation of basic molecules while reversed phase LC was preferred for the resolution of acids. SFC showed the broadest enantioselectivity overall and normal phase LC indicated similar properties, as expected, to SFC but with lower column efficiency.

63

Title: Effect of sample solvents on

retention in packed column supercritical fluid chromatography (Article in Chinese)

Authors: Lu F, Liu LL, Wu YT

Journal: Se Pu. 2000 18(2) 155-157

Address: Second Military Medical University, College of Pharmacy, Shanghai 200433, China.

Abstract: Factors affecting the retention in supercritical fluid chromatography(SFC) on C18 and NH2 packed columns have been studied. These factors include the sampling solvent, continuous injection, stationary phase type and concentration of modifier. When supercritical carbon dioxide or low concentration(less than 0.5%) of methanol is employed as the mobile phase, retention time increases with the polarity of the solvent on NH2 column, while it keeps almost constant on C18 column. Methanol will cover the active sites on the surface of the bonded phase, forming the "pseudo" stationary phase, which will lower the interaction between the analytes and the stationary phase, hence the shorter will be the retention time. The hydrogen bond between stationary phase (including the amino group and the remaining hydroxyl group) and methanol is the most obvious. So it's quite more evident on NH2 column than on C18 column that the delaying effect of the solvent, with methanol, acetone and chloroform in decreasing sequence, encourages the elution of the analytes during continuous injections. The elution will recover to the first injection after adequate interval has passed, i.e., more than 10 column volumes. When the methanol concentration is greater than 1.0% in carbon dioxide, the effect decreases and becomes negligible. The solvent effect is significant to the reasonable injection and reproducible chromatographic data in packed column SFC.

Title: Separation of metal chelates and

organometallic compounds by SFC and SFE/GC.

Authors: Wai CM, Wang S


Address: University of Idaho, Department of Chemistry, Moscow, ID 83844-2343, USA.

Abstract: Supercritical fluid chromatography (SFC) combines the high diffusion coefficients of gas chromatography (GC) and the solubility properties of liquid chromatography (LC). SFC generally requires lower temperatures for chromatographic separations and thus is more suitable for analyzing thermally labile compounds including a number of metal chelates and organometallic compounds. SFC also allows interfacing between supercritical fluid extraction (SFE) and chromatographic analysis of metal-containing compounds. A large number of metal chelates and organometallic compounds can be separated by SFC. This article summarizes SFC separation of various chelates of transition metals, heavy metals, lanthanides and actinides as well as organometallic compounds of lead, mercury, and tin reported in the recent literature. This article also discusses SFC detection systems and the determination of solubility of organometallic compounds by SFC.

Title: Determination of the 2-

bromomethyl-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methyl benzoate diastereoisomers by supercritical fluid chromatography

Authors: Toribio L, Bernal JL, del Nozal MJ, Jimenez JJ, Serna ML

Journal: J Biochem Biophys Methods. 2000 43 251-260

Address: University of Valladolid, Department of Analytical Chemistry, Faculty of Sciences, Prado de la Magdalena s /n, E-47005 Valladolid, Spain

Abstract: The study of the separation and determination of the two 2-bromomethyl-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl-methyl benzoate diastereoisomers is presented in this work. The separation was achieved using packed column supercritical fluid chromatography. Three stationary phases were checked, the C18 packing providing the best results. The influence of several 18 parameters (nature and percentage of modifier, temperature and pressure) on the separation were also evaluated. The baseline separation (R 51.78) of the compounds was obtained in only 2 mins, using a C18 column, a pressure of 100 bar, a temperature of 358C, a flow-rate of 2.5 ml/min and 18 5% of 2-propanol as organic modifier. Under these conditions the unwanted diastereoisomer (trans) was eluted before the main compound (cis), so the determination of the purity was more accurate.

Title: Enantiomer separation by

complexation SFC on immobilized Chirasil-nickel and Chirasil-zinc.

Authors: Schurig V, Fluck M


Address: Institute of Organic Chemistry, University of Tubingen, Auf der Morgenstelle 18, 72076, Tubingen, Germany.

Abstract: The use of complexation SFC for enantiomer separation of Lewis base selectands on chiral nickel(II)- and zinc(II)-bis[(3-heptafluorobutanoyl)-10-methylene-(1R)-camphora te] chemically bonded to poly(dimethylsiloxane) (Chirasil-nickel and Chirasil-zinc) and employed as Lewis acid selectors is described. The method is especially suited for less volatile and configurationally labile racemates. The variation of the experimental parameters temperature T, pressure p and density rho of the mobile phase carbon dioxide on the retention factor k, relative retention r and chiral separation factor alpha is studied, providing insights into the mechanisms of chiral recognition under supercritical conditions. For mecoprop methyl ester (methyl 2-(4-chloro-2-methylphenoxy)propanoate) an unusual increase of alpha at increased temperature is observed on Chirasil-nickel. Supercritical carbon dioxide does not inadvertently affect the complexation equilibria between Lewis donor selectands and the Lewis acid metal selectors during complexation SFC.

65

Title: SFC/FTIR, SFC/APCI-MS and

MALDI-TOF-MS for the analysis of siloxane-ethylene oxide copolymers.

Authors: Just U, Jones DJ, Auerbach RH, Davidson G, Kappler K


Address: Federal Institute for Materials Research and Testing (BAM), Fachgruppe VI.3, Unter den Eichen 87, 12200, Berlin, Germany.

Abstract: Polydimethylsiloxanes (PDMSs) modified by introducing ethylene oxide units with the aim of forming sufficiently water-soluble siloxane compounds were characterized using supercritical fluid chromatography (SFC) coupled with Fourier transform infrared (FTIR) spectroscopy and atmospheric pressure chemical ionization mass spectrometry (APCI-MS), and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS). SFC has a domain in analyzing oligomers. Hyphenated techniques enable elucidation of the components. Remarkable is the resolution and short analysis time of MALDI-TOF-MS. SFC also allows quantification of the basic and reaction products.

Title: Carbon-based quantitation of

pyrethrins by supercritical-fluid chromatography.

Authors: Wenclawiak B, Otterbach A


Address: University of Siegen, Department of Analytical Chemistry, Adolf-Reichwein-Strasse 2, 57068, Siegen, Germany.

Abstract: All six insecticide active ingredients in pyrethrum extract were quantified by supercritical fluid chromatography and carbon calibration. Allethrin is a suitable reference compound for carbon calibration and pyrethrins calibrations. Carbon quantification in SFC is also applied to pyrethroids (phenothrin, permethrin, cypermethrin, fenvalerate and deltamethrin) and alkanes. Halogen substitution on pyrethroids requires halogens on the reference calibration compound. The method was applied to commercial extracts.


as basis for identification and quantitative determination of

Abstract: Indol-3-ylmethylglucosinolate (glucobrassicin) occurs in most plants of the Brassicaceae family together with hydroxy and methoxy derivatives of glucobrassicin. These compounds and products

indol-3-ylmethyl oligomers and ascorbigens.

Authors: Buskov S, Olsen CE, Sorensen H, Sorensen S


Address: Royal Veterinary and Agricultural University, Chemistry Department, 40 Thorvaldsensvej, DK-1871 C, Frederiksberg, Denmark

produced therefrom have been the subject of considerable research interest due to their potential anticarcinogenic effects, and thereby a need for techniques to work with the individual compounds. A method using normal-phase supercritical fluid chromatography (SFC) with methanol as modifier has been developed for determination and quantification of the various indol-3-ylmethyl derivatives including ascorbigens formed from the glucobrassicin degradation product, indol-3-ylmethanol, under acidic conditions (pH 2-6) with and without the presence of ascorbic acid. The SFC method had detection limits in the 10-100-pmol range. In the absence of ascorbic acid a range of oligomers were formed, whereas the presence of ascorbic acid favoured the formation of ascorbigen and products thereof. Quantitatively important indol-3-ylmethyl oligomers consisting of up to five indol rings have been purified with preparative SFC and identified from MS and 1D and 2D NMR experiments with complete assignment of chemical shifts to all of the atoms. Investigation of the autolysis products of white cabbage showed that ascorbigens were the quantitatively dominating degradation products of indol-3-ylmethylglucosinolates.


as a method of analysis for the determination of 4-hydroxybenzylglucosinolate degradation products.

Authors: Buskov S, Hasselstrom J, Olsen CE, Sorensen H, Sorensen JC, Sorensen S


Address: Royal Veterinary and Agricultural University, Chemistry Department, 40 Thorvaldsensvej, DK-1871 C, Frederiksberg, Denmark

Abstract: In the present study analytical and preparative supercritical fluid chromatography (SFC) were used for investigation of myrosinase catalysed degradation of 4-hydroxybenzylglucosinolate (sinalbin). Sinalbin occurs as a major glucosinolate in seeds of Sinapis alba L., in various mustards and other food products. The degradation products were identified and quantified by analysis based on a developed SFC method using a bare silica column. Determinations comprised transformation products of sinalbin, produced both during degradation of isolated sinalbin, and during autolysis of meal from S. alba seeds. The conditions in the developed SFC method were used as basis for the preparative SFC procedure applied for isolation of the components prior to their identification by nuclear magnetic resonance (NMR) spectroscopy. Myrosinase catalysed sinalbin hydrolysis resulted in the reactive 4-hydroxybenzyl isothiocyanate as an initial product at pH values from 3.5 to 7.5 whereas 4-hydroxybenzyl cyanide was one of the major products at low pH values. 4-Hydroxybenzyl isothiocyanate was found to disappear from the aqueous reaction mixtures in a few hours, as it reacted easily with available nucleophilic reagents. 4-Hydroxybenzyl alcohol was found as the product from reaction with water, and with ascorbic acid, 4-hydroxybenzylascorbigen was produced.

67

Title: Separation of vitamins by

supercritical fluid chromatography with water-modified carbon dioxide as the mobile phase.

Authors: Pyo D


Address: Kangwon National University, Department of Chemistry, 200-701, Chuncheon, South Korea.

Abstract: Supercritical fluid chromatography (SFC) has become a technique for solving problems that are difficult to be monitored by other chromatographic methods. However, the most widely used fluid, is no more polar than hexane. Polar samples which are difficult to be analyzed with pure supercritical CO(2) because of their high polarity can be separated by adding polar modifiers to supercritical CO(2). In this paper various vitamins were well separated using water-modified supercritical CO(2) fluid. The amount of water dissolved in supercritical CO(2) was measured using an amperometric microsensor made of a thin film of perfluorosulfonate ionomer (PFSI).

Title: The development of a semi-

preparatory scale supercritical-fluid chromatograph for high-throughput purification of 'combi-chem' libraries.

Authors: Berger TA, Fogleman K, Staats T, Bente P, Crocket I, Farrell W, Osonubi M.


Address: Berger Instruments, 123A Sandy Drive, Newark, DE 19713, USA.

Abstract: A unique separator was developed which allowed automatic separation and peak collection using semi-preparative supercritical fluid chromatography (SFC). A peak detector switched the effluent between waste and special collection cassettes. Up to 50 mg of various solutes were injected onto a 21-mm I.D. Cyano column. The entire flow path was contained and no aerosols were generated. Collection efficiency was as high as 95%. Peak purity was often greater than 99. 9%. Typical run times were less than 10 min. An analytical SFC was used to screen the performance of a wide range of mobile and stationary phases for the elution of more than 60 miscellaneous small drug compounds. The best 'universal' gradient employed 0.4% isobutyl or isopropylamine dissolved in methanol, then mixed from 5 to 55% into carbon dioxide at 10%/min. Flow rate was 50 ml/min. The analytical SFC was shown to be a good predictor of the semi-prep instrumental performance.

Title: High-speed screening of

combinatorial libraries by gradient Abstract: Packed-column supercritical fluid chromatography (pSFC) with a fast composition gradient is used

packed-column supercritical fluid chromatography

Authors: Berger TA, Wilson WH

Journal: J Biochem Biophys Methods. 2000 43 77-85

Address: Berger Instruments, 123A Sandy Drive, Newark, DE 19713, USA.

as a rapid screening tool for combinatorial chemistry. The advantages of fast analysis speed and fast recovery to initial conditions are demonstrated. Retention time reproducibility is similar to isocratic analyses by pSFC and ranges from 0.37 to 0.64% relative standard deviation. A mixture of b-blockers illustrates the ‘normal phase’ retention mechanism. For these solutes and a range of analytes, the peak width is relatively constant. Such behavior permits the classical peak capacity equation to be reduced to a simple, straightforward form. Using this as a performance metric, pSFC is shown to be 5–10 times faster than reversed phase HPLC for library screening.

Title: Separation of drugs by packed-

column supercritical fluid chromatography

Authors: Yakua K, Morishitab F


Address: Tanabe Seiyaku Co. Ltd., Analytical Chemistry Department, Product & Technology Development Laboratory, 16-89 Kashima 3-chome, Yodogawa-ku, Osaka 532-8505, Japan

Abstract: Packed-column supercritical fluid chromatography (pSFC) has been expected to analyze various kinds of compounds. Many researchers have expected a new chromatographic technique that overcomes the limitations of other techniques, HPLC and GC. In pharmaceutical development, chromatography plays an important role in the evaluation of safety and efficacy of a new compound. This article provides an overview of the separation of drugs by pSFC. The effects of the chromatographic parameters were studied for the separation of steroids. In chiral separation, the successful results were shown and compared with HPLC.

Title: Element selective detection for

supercritical-fluid chromatography.

Authors: Vela NP, Caruso JA

Journal: J Biochem Biophys Methods. 2000

Abstract: This manuscript describes the use of Supercritical-Fluid Chromatography (SFC) with plasma spectrometric detection for the analysis of organometallics. An introduction on the principles and characteristics of Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) and Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) is included, along with a discussion

69

43(1-3) 45-58

Address: US Food and Drug Administration, Forensic Chemistry Center, 6751 Steger Drive, Cincinnati, OH 45237, USA.

about requirements for coupling SFC to plasma detection and the different approaches for interfacing SFC to ICP. The last part of this review paper provides a comprehensive description of SFC-ICP applications for the analysis of organometallics containing iron, silicon, tin, chromium, arsenic, lead, mercury and antimony.

Title: Tuning of mobile and stationary

phase polarity for the separation of polar compounds by SFC.

Authors: Ibanez E, Senorans FJ


Address: Instituto de Fermentaciones Industriales, C.S.I.C., Juan de la Cierva 3, 28006 Madrid, Spain.

Abstract: The separation of polar compounds by supercritical-fluid chromatography (SFC) is reviewed. New developments in mobile and stationary phase tuning are reviewed for packed and packed capillary SFC. In terms of mobile phase polarity adjustment, new pure and multiple component fluids are presented. The approach of tuning the polarity of the stationary phase as a way of increasing the range of polar compounds analyzed by SFC using pure CO(2) is discussed using either silica-based or new materials as stationary phase. Chiral, liquid crystal and polymer-based stationary phases coated on particles are widely covered in this review as an interesting approach to separate polar compounds avoiding the major drawbacks associated to the use of modifiers in SFC.

Title: Progress in packed column

supercritical fluid chromatography: materials and methods.

Authors: Poole CF


Address: Wayne State University, Department of Chemistry, Rm. 171 Chemistry, Detroit, MI 48202, USA.

Abstract: This article summarizes recent developments in packed column supercritical fluid chromatography. Silica-based chemically bonded sorbents, similar to those used for HPLC, are widely used with solvent-modified fluids containing additives to suppress undesirable solute-sorbent interactions that lead to poor peak shapes. Composition programming is the most useful approach to gradient elution separations since solvent-modified fluids have low compressibility. Packed column SFC is most useful for the separation of mixtures usually separated by normal-phase HPLC. Compared to normal-phase HPLC it offers faster separations, higher efficiencies, faster column re-equilibration, and a wider range of experimental variables for optimization. Packed column SFC is being increasingly selected for the analytical and preparative separation of racemic mixtures using enantiomer-selective sorbents.

Title: SFC of drug enantiomers.

Authors: Phinney KW

Journal: Anal Chem. 2000 72(5) 204A-211A.

Address: National Institute of Standards and Technology, Gaithersburg, MD 20899-8392, USA.

Abstract:

Title: Separation of divalent transition

metal b-diketonates and their adducts by supercritical fluid chromatography.

Authors: Lin Y, Wu H, Wai CM, Smart NG

Journal: Talanta 2000 52 695–701

Address: Pacific Northwest National Laboratory, Environmental Molecular Sciences Laboratory, PO Box 999, MS: K8 -93, Richland, WA 99352, USA

Abstract: A method for separation and detection of divalent transition metal b-diketonates by adduct formation:supercritical fluid chromatography (SFC) with an open-tubular capillary column and a FID detector is described. The crystal structures of copper (Cu)-hexafluoro-acetylacetone (HFA) and Cu bis(2,2,6,6-tetramethyl-3,5-heptanedionato) (THD) complexes have been determined by X-ray crystallography. The SFC behavior of Cu b-diketonates shows a strong correlation with the structure of the complexes. The hydrated cu b-diketonate complexes usually exhibit strong intermolecular interactions or decomposition in SFC. Formation of adducts with a neutral donor, such as tributyl phosphine oxide (TBPO), can greatly improve the SFC behavior and detection sensitivity of Cu(II) and Mn(II) b-diketonates. The stoichiometry and thermal stability of the adducts Cu(II) and Mn(II) b-diketonates with TBPO in supercritical CO2 have also been investigated. The implications of utilizing adduct formation for supercritical fluid extraction (SFE) of divalent transition metals and for on-line coupled SFE:SFC analysis of divalent transition metals are discussed

Title: The analysis of beta-agonists by

packed-column supercritical fluid chromatography with ultra-violet and atmospheric pressure chemical ionisation mass spectrometric detection.

Abstract: Packed-column supercritical fluid chromatography (pSFC) using ultra-violet (UV) and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS) provides a versatile method for the identification and quantification of beta-agonists. We have achieved good separation of clenbuterol, salbutamol, terbutaline and fenoterol with good resolution and reasonable retention times using a high concentration of methanol modifier in the supercritical CO2, together with small amounts of

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Authors: Jones DC, Dost K, Davidson G, George MW

Journal: Analyst. 1999 124(6) 827-831

Address: University of Nottingham, School of Chemistry, UK.

both acidic (trifluoroacetic acid, TFAA) and basic (triethylamine, TEA, or diethylamine, DEA) additives. APCI-MS gave unambiguous identification of the 4 analytes, and increasing cone voltage provided informative fragmentation patterns. The pSFC-MS technique was shown to be linear (R2 > or = 0.996) over the concentration range 1-50 micrograms ml-1. Single ion monitoring (SIM) gave detection limits (on-column) of 2.5 ng (clenbuterol), 0.83 ng (terbutaline), 7.6 ng (salbutamol) and 2.7 ng (fenoterol). The pSFC-MS system was shown to be reproducible within a day, between days, and between restrictors. Analysis of milk samples 'spiked' with beta-agonists showed that the matrix caused no interference, with detection limits of approximately 500 micrograms l-1 of beta-agonists. More dilute solutions could be analysed by pre-concentration before the SFC stage.

Title: Optimization of the extraction and

fractionation of corn bran oil using analytical supercritical fluid instrumentation.

Authors: Taylor SL, King JW


Address: Food Quality and Safety Research Unit, U.S. Department of Agriculture, Peoria, IL 61604, USA.

Abstract: Supercritical fluid extraction (SFE) is combined with supercritical fluid chromatography (SFC) in an analytical mode to develop a system for fractionating and enriching high value ferulate-phytosterol esters (FPE) contained in corn bran oil. Corn bran is initially extracted with neat supercritical carbon dioxide (SC-CO2) at various pressures (13.8, 34.5, and 69 MPa) and temperatures (40, 60, and 80 °C) to see if the FPE can be enriched in the extracts. These initial studies show the greatest percentage of FPE could be extracted under two sets of conditions: 69 MPa at 80°C and 34.5 MPa at 40°C. Both sets of parameters yield an extract containing approximately 1.25% FPE. A stock supply of corn bran oil is then produced by scaled-up SFE at 34.5 MPa and 40 °C for subsequent chromatographic fractionation. The SFE-obtained corn bran oil is then applied to the head of a minichromatographic column containing an amino-propyl sorbent. SFC is than commenced using neat SC-CO2 at 69 MPa and 80°C to remove the majority of the triglyceride-based oil. Pressure and temperature are then lowered to 34.5 MPa and 40°C, respectively, and ethanol is added as a modifier. The modifier is added in an increasing stepwise gradient program, and fractions are collected at equal volume intervals. The resultant fractions are analyzed by analytical high-performance liquid chromatography with evaporative light-scattering detection and show that FPE could be enriched to a 14.5% (w) level.

Title: Feasibility of lovastatin analysis

by packed column supercritical Abstract: A reliable supercritical fluid chromatography (SFC) method was developed for the analysis of

fluid chromatography with ultraviolet detection.

Authors: Strode JT, Taylor LT, Howard AL, Ip D


Address: Virginia Polytechnic Institute and State University, College of Arts and Sciences, Department of Chemistry, Blacksburg 24061-0212, USA.

lovastatin, a hypocholesterolaemic drug, from MEVACOR. Methanol-modified carbon dioxide was shown to elute the drug, and its dehydrolovastatin and hydroxy acid lovastatin degradation products from a Hypersil silica column. However, the hydroxy acid lovastatin was found to tail in this mobile phase. The phenomena was eliminated by the addition of trifluoroacetic acid [Haouck, S. Thomas, D. K. Ellison, Talanta 40 (1993) 491] to the mobile phase which permitted the drug and its two main degradation products to all elute from the Hypersil silica column in under 6 min with symmetrical peak shape. Chromatographic limit of detection (LOD) and limit of quantification (LOQ), linear dynamic range (LDR), and injection precision were obtained in order to assess the chromatographic performance of the SFC system for the lovastatin separation.

Title: Separation of cis/trans isomers of

b-carotene by supercritical fluid chromatography

Authors: Lesellier E, Gurdale K, Tchapla A


Address: Groupe de chimie analytique de l'Universite Paris Sud (XI), L.E.T.I.A.M.. IUT ORSAY, Plateau de Moulon, 91400 Orsay, France.

Abstract: The aim of this work was to study and improve the separation of b-carotene cis /trans isomers in SFC. Five different stationary phases have been tested, and their chromatographic behaviour has been determined. Unfortunately, because of the presence of additional isomers in the sample studied, none of the individual columns were able to isolate the four pure main isomers of b-carotene (9, 13, 15 cis and trans). The coupling of two different monomeric octadecyl columns was necessary to increase both selectivity and efficiency. The low viscosities of supercritical mobile phases allowed this column association. The influence of temperature, pressure, nature and modifier content were also investigated. Finally, with the optimum chromatographic conditions, six additional isomers have been separated from the four previous ones. One of them has been identified as the 9–99 di-cis b-carotene.

Title: Separation of stereoisomers in a

simulated moving bed-supercritical fluid chromatography plant.

Authors: Depta A, Giese T, Johannsen M, Brunner G

Abstract: The combination of two techniques, simulated moving bed (SMB) and supercritical fluid chromatography (SFC), leads to an apparatus with unique features. Besides the known advantages of the SMB process, like reduced solvent consumption and its continuity, the use of supercritical carbon dioxide as the mobile phase offers an easy product recovery by depressurizing the

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Address: Technische Universitat Hamburg-Harburg, Arbeitsbereich Verfahrenstechnik II, Germany.

supercritical fluid. Details of a SMB-SFC plant are presented for the first time. Due to the large number of process parameters a simulation of the SMB process is necessary to achieve optimal operating conditions. The most important thermodynamic information for a SMB process is the adsorption isotherms. Therefore, isotherms for two phytol isomers are measured and correlated. A fast dynamic model for the simulation of SMB is used to calculate the region of complete separation taking different column configurations and the compressibility of the mobile phase into account.

Title: Direct connection of supercritical

fluid extraction and supercritical fluid chromatography as a rapid quantitative method for capsaicinoids in placentas of Capsicum.

Authors: Sato K, Sasaki SS, Goda Y, Yamada T, Nunomura O, Ishikawa K, Maitani T

Journal: J Agric Food Chem. 1999 47(11) 4665-4668

Address: National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya, Tokyo 158-8501, Japan.

Abstract: The fruits of Capsicum annuum L. are used worldwide as chili peppers and in folk medicines. The pungent components of C. annuum, which are irritants, are called capsaicinoids (CAPS), and the most abundant components are capsaicin, dihydrocapsaicin, and nordihydrocapsaicin. To analyze CAPS in the placentas of Capsicum fruits rapidly and safely, we used a directly connected system of supercritical fluid extraction and supercritical fluid chromatography (SFE/SFC). As a column for SFE/SFC, only a silica-type column was found to be suitable. The CAPS contents in placentas of C. annuum cv. Jalapeno (hot type) and C. annuum cv. Shishitoh (less-hot type) determined by the SFE/SFC method agreed well with those in the range of 0-13.81 mg g(-1) fr. wt determined by the usual extraction-HPLC method. The SFE/SFC method has the advantages of no need for pretreatment and no (or minimal) need for organic solvents. We conclude that this method is useful as a rapid (20 min) and safe screening test for the pungency of various Capsicum fruits.

Title: Potential of packed column

supercritical fluid chromatography for the separation of metoprolol from closely related compounds.

Authors: Gyllenhaal O, Vessman J


Abstract: Using the present SFC method metoprolol can be separated from 12 analogues and related compounds within 12 min. It is also possible to detect most of these analogues at the 0.1% (w/w) level when added to the pure drug substance. The method is based on SFC with modified carbon dioxide as the mobile phase and a packed 4 mm I.D. diol-modified silica column. The mobile phase modifier is 10% (v/v) methanol containing 0.35 M of acetic acid and 0.07 M of triethylamine. UV detection is performed at 273 nm. The main advantage with the described method

Address: Astra Hassle AB Analytical Chemistry, Pharmaceutical R & D, S-431 83 Molndal, Sweden

over the currently used reversed-phase liquid chromatographic method is the different selectivity, i.e., it is possible to separate and detect isomers that elute close to the metoprolol peak in currently used liquid chromatographic systems.

Title: Comparison of methods for

quantitative analysis of additives in low-density polyethylene using supercritical fluid and enhanced solvent extraction.

Authors: Zhou LY, Ashraf-Khorassani M, Taylor LT


Address: Virginia Tech, Department of Chemistry, Blacksburg 24061-0212, USA.

Abstract: On-line supercritical fluid extraction-supercritical fluid chromatography (SFE-SFC) with cryogenic trapping was used to extract and separate five additives from a low-density polyethylene (LDPE) sample. A glass tube filled with glass wool afforded excellent collection efficiency for the extracted analytes. Additive spiked sand was employed to optimize the various parameters of the on-line SFE-SFC system. Calibration curves from the spiked sand studies for on-line SFE-SFC were obtained with good linearities for quantitation. Results obtained on additives in LDPE from on-line SFE-SFC were comparable to those from off-line SFE-HPLC and off-line enhanced solvent extraction (ESE)-HPLC for all additives except Irganox 1076. However, the precision obtained with on-line SFE-SFC was lower than that from off-line SFE-HPLC and off-line ESE-HPLC due to the small sample size employed in the on-line system. Considerable clean-up of the ESE extract was required prior to chromatographic analysis. On-line SFE-SFC minimized the sample handling and eliminated the use of organic solvent. Despite the lower than expected precision, the on-line SFE-SFC method for quantitation of polymer additives appears to be reliable and robust for application in routine quality control analysis.

Title: Supercritical fluids in separation

science – the dreams, the reality and the future.

Authors: Smith RM

Journal: J. Chromatogr. A 1999 856 83 –115

Address: Loughborough University, Department of Chemistry,

Abstract: The last 20 years have seen an intense interest in the use of supercritical fluids in separation science. This started with the introduction of commercial instruments first for packed and then for capillary chromatography and it looked as if this would be a technique to rival gas–liquid chromatography and HPLC. The activity developed quite rapidly into packed column supercritical fluid separations then into supercritical fluid extraction. However, in recent years there has been a decline in publications. These later techniques continue to be used but are now principally applied to a limited group of applications where they offer significant advantages over alternative techniques. This review looks back over this period and analyses how these methods were developed and the

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Loughborough, Leics. LE11 3TU, UK

fluids, detectors and applications that were examined. It suggests why many of the initial applications have vanished and why the initial apparent promise was not fulfilled. The rise and fall of supercritical fluids represents a lesson in the way analysts approach new techniques and how we might view other new separation developments at the end of this millennium. The review looks forward to the future of supercritical fluids and their role at the end of the first century of separation science. Probably the most important idea that supercritical fluids have brought to separation science is a recognition that there is unity in the separation methods and that a continuum exists from gases to liquids.

Title: Electrospray mass spectrometry

and supercritical fluid chromatography of methylated beta-cyclodextrins.

Authors: Salvador A, Herbreteau B, Dreux M


Address: Institut de Chimie Organique et Analytique (ICOA), UPRES A, Universite d'Orleans, France.

Abstract: Five commercial dimethylated beta-cyclodextrin (DM-beta-CD) samples were analysed by electrospray (or ionspray) mass spectrometry (ESI-MS) and supercritical fluid chromatography (SFC) with evaporative light scattering detection. A silica and a nitro-bonded silica were selected using CO2-methanol-acetonitrile-water and CO2-methanol as mobile phase, respectively. An extensive optimisation scheme was performed for mobile phase selection. Both SFC systems were used for analyses of complex DM-beta-CD samples. Peak identifications were made using off-line ESI-MS. Commercial DM-beta-CDs are impure mixtures of homologues and isomers and analysis reveals that every manufacturer produces a different mixture.

Title: Chiral drug separation by

supercritical fluid chromatography (Article in Chinese)

Authors: Li H, Hu X

Journal: Se Pu. 1999 17(2) 166-170

Address: Wuhan University,

Abstract: Because of the high-efficiency, fast separation, low-temperature analysis and applicable to a wide variety of detectors, supercritical fluid chromatography(SFC) is an attractive alternative for chiral drug separation. The development and application of this technique in enantiomer separation are reviewed with 48 references. The separation modes and the choices of experimental condition are discussed.

Department of the Science of Analysis and Measurement, Wuhan, 430072, China.

Title: Recent innovations in enantiomer

separation by electrochromatography utilizing modified cyclodextrins as stationary phases.

Authors: Schurig V, Wistuba D

Journal: Electrophoresis. 1999 20(12) 2313-2328

Address: Institut fur Organische Chemie, Universitat Tubingen, Germany.

Abstract: Enantiomer separation by electrochromatography employing modified cyclodextrins as stationary phases is performed in two ways. (i) Polysiloxane-linked permethylated beta-cyclodextrin (Chirasil-Dex 1) or related selectors are coated and immobilized onto the inner surface of a capillary column. Enantiomer separation is performed in the open tube and the method is referred to as open-tubular capillary electrochromatography (o-CEC). (ii) Silica-linked native beta-cyclodextrin, permethylated beta-cyclodextrin (Chira-Dex 2) or hydroxypropyl-beta-cyclodextrin are filled into a capillary column and the bed is secured by two frits. Enantiomer separation is performed in a packed column and the method is referred to as packed capillary electrochromatography (p-CEC). In a unified instrumental approach, method (i) as well as method (ii) can be operated both in the electro- and pressure-driven modes (o-CEC vs. open-tubular liquid chromatography (o-LC) and p-CEC vs. p-LC). It is demonstrated that the electro-driven variant affords higher efficiencies at comparable elution times. Employing a single open-tubular column coated with Chirasil-Dex 1, a unified enantioselective approach can be realized in which the same selectand is separated using all existing chromatographic modes for enantiomers, i.e., gas chromatography (GC), super-critical fluid chromatography (SFC), o-LC and o-CEC. As the chiral selector is utilized as a stationary phase, an additional chiral selector may be added to the mobile phase. In the resulting dual chiral recognition systems, enhancement of enantioselectivity (matched case) or compensation of enantioselectivity (mismatched case) may be observed. The overall enantioselectivity is dependent on the sense of enantioselectivity of the selectors chosen and their influence on the electrophoretic and electroosmotic migration of the enantiomers of a selectand.


in forensic science: a critical appraisal.

Authors: McAvoy Y, Backstrom B, Janhunen K,

Abstract: The application of supercritical fluid chromatography (SFC) in forensic science is reviewed. The applications centre on the analysis of explosives and of drugs of abuse. The systems employed are discussed in the context of comparison with gas chromatography and high-performance liquid

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Stewart A, Cole MD Journal: Forensic Sci Int. 1999

99(2) 107-122

Address: University of Stratholyde, Department of Pure and Applied Chemistry, Glasgow, United Kingdom

chromatography methods which are traditionally used for such analyses. The advantages and disadvantages of SFC over these methods are discussed. Recommendations are made for the developments which are required in SFC technology if it is to find greater application in forensic science.

Title: Analysis of amphetamines by

supercritical fluid chromatography, high-performance liquid chromatography, gas chromatography and capillary zone electrophoresis - a preliminary comparison

Authors: McAvoy Y, Gueniat O, Cole MD

Journal: Forensic Sci Int. 1999 103 13-22

Address: University of Stratholyde, Forensic Science Unit, Department of Pure and Applied Chemistry, 204 George Street, Glasgow G1 1XW, United Kingdom

Abstract: A supercritical fluid chromatographic method with UV detection (SFC–UV) for the quantitative separation of phenylisothiocyanate (PITC)-derivatised amphetamines is described and compared to high-performance liquid chromatography–diode array detection (HPLC–DAD), gas chromatography– flame ionisation detection (GC–FID) and capillary zone electrophoresis–diode array detection (CZE–DAD) analyses of amphetamine and related compounds. Difficulties in the analysis of common amphetamines by SFC are discussed. Of the methods described in this paper, the SFC method offered the greatest sensitivity at 0.01–0.02 mg of drug on column. Suggestions are made for the use of combinations of these techniques for the identification of amphetamines when gas chromatography–mass spectrometry is not available.

Title: Application of packed column

supercritical fluid chromatography Abstract: Studies of speed, resolution, and selectivity have shown that packed column supercritical fluid chromatography (PCSFC) is a viable technique for the isocratic, isothermal and isobaric separation

to the simultaneous determination of seven anticonvulsant drugs

Authors: Patil ST, Sundaresan M, Bhoir IC

Journal: Talanta 1998 48 1179-1189

Address: Patel Research Centre For Chemistry and Biological Sciences, Mithibai College Building, Vile Parle (West), Mumbai 400 056, India

of seven anticonvulsants, viz., phenobarbitone, phenytoin sodium, phethenylate sodium, nitrazepam, clonazepam, carbamazepine, and primidone, and their simultaneous estimation. The drugs were eluted from a JASCO, RP-C18 (250 4.6 mm) 10 m packed column with a binary mobile phase of carbon dioxide and methanol, using ibuprofen as the internal standard. The effect of pressure, temperature, modifier concentration, and the rate of flow of CO2 on retention and selectivity of all the analytes were studied and the parameters optimised. Without methanol in the mobile phase none of the solutes eluted. Changing modifier concentration was the most effective physical parameter for changing retention and selectivity. The analytes were detected using a UV detector at 215 nm. An arbitrary mixture of eight components was baseline resolved in 7 min. The study includes a successful attempt at quantification of the drugs. Chromatographic and analytical figures of merit have been listed. The present work holds promise for a possible replacement of HPLC with SFC for the separation and assay of drugs of different families.

Title: Determination of androstenone in

pig fat using packed column supercritical fluid chromatography-mass spectrometry.

Authors: Tuomola M, Hakala M, Manninen P

Journal: J Chromatogr B Biomed Sci Appl. 1998 719(1-2) 25-30

Address: University of Turku, Department of Biochemistry and Food Chemistry, Finland

Abstract: Packed column supercritical fluid chromatography (SFC) in combination with atmospheric pressure chemical ionisation mass spectrometry was applied to the analysis of androstenone in pig fat samples. Liquefied fat samples were dissolved in dichloromethane and analysed directly by SFC without any sample purification. Chromatographic separation was achieved with a density/pressure gradient using pure carbon dioxide as the mobile phase and the analysis resulted in a quantitation limit of 0.25 microg/g with 1 microl injection volume. Good agreement was found between the SFC method and time-resolved fluoroimmunoassay by the analysis of 15 boar back fat samples.

Title: Analytical supercritical fluid

chromatography using fully Abstract: Automated analytical supercritical fluid chromatography is used to separate enantiomers of

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automated column and modifier selection valves for the rapid development of chiral separations.

Authors: Villeneuve MS, Anderegg RJ

Journal: J. Chromatogr. A 1998 826 217-225

Address: GlaxoWellcome Inc., Analytical Chemistry, Five Moore Drive, Research Triangle Park, NC 27709, USA

pharmaceutical compounds in the drug discovery laboratory. Modification of a commercial instrument to incorporate a six-way column selection valve, multiple chiral columns based on derivatized cellulose or amylose, and a four-way modifier selection valve provides a powerful combination for the rapid development of chiral separations. A wide set of columns and conditions can be tested sequentially, including unattended operation overnight. This paper shows that similar racemic compounds, even those from the same molecular class, are separated using different column and modifier combinations. Therefore, the use of program-controlled column and modifier selectors has great advantages. Using the fully automated system, the optimal chiral separation of several compounds can be obtained unattended within 24 h.


chromatography coupled with solid-phase extraction for the determination of organic microcontaminants in water.

Authors: Toribio L, del Nozal MJ, Bernal JL, Jimenez JJ, Serna ML



Abstract: A multiresidue method for the analysis of 35 common contaminants (including pesticides, polycyclic aromatic hydrocarbons and phenols) in lough and river waters from Castilla y Leon Spanish region, by using supercritical fluid chromatography (SFC) with five silica packed columns, is described. In order to decrease the detection limits, a preconcentration step by coupling solid-phase extraction to the SFC system was used. The different variables affecting the extraction procedure were studied and optimized, selecting the Isolute Env+ as the best sorbent. The detection limits achieved ranged from 0.4 to 2.6 micrograms l-1.

Title: Lipid uptake by silicone enteral

access feeding devices. Abstract: Deterioration of long-term enteral access devices remains a significant patient care and financial

Authors: Trudel J, Gauderer MW, Drews MJ, LaBerge M

Journal: J Pediatr Surg. 1998 33(6) 880-884

Address: Greenville Hospital System/Clemson University Biomedical Cooperative, Department of Bioengineering and School of Textile, Fiber, and Polymer Science, Clemson University, South Carolina, USA

problem. Because it is known that lipids, particularly medium-chain triglycerides oil (MCT oil), can soften and break certain types of polymeric materials, the authors evaluated the effect of liquid enteral feeding formulas containing different amounts of MCT oil on silicone feeding tubes.Commercially available and widely used gastrostomy silicone catheters of two sizes (20F and 15F) were sectioned in 5-cm-long samples and cleaned. Five groups of five pieces were immersed for 8 weeks in pure MCT oil and in four commercial formulas having similar protein, carbohydrate, and fat contents. These formulas were chosen because they contained similar percentages of fat (37% to 45%), although they contained differing amounts of MCT oil. Sodium azide 0.05% was added as a bactericide. Samples were maintained at 37 degrees C in an incubator-shaker. After incubation, specimens were rinsed, dried, and weighed. Selected samples were extracted with a chloroform-methanol solution (2:1). Super-critical fluid chromatography (SFC) and infrared (IR) spectroscopy were performed. A tensile tester was used to generate force-elongation curves for the remaining samples. A statistical analysis (ANOVA, alpha = .05) was conducted to compare data from test groups with results from 20 samples of control silicone material. Data demonstrate that silicone is significantly affected by liquid formulas and pure MCT oil. SFC and IR findings indicated that fractions of MCT oil, corn oil, and canola oil were absorbed by the material. The most dramatic weight gain (3.7%) was observed for specimens immersed in pure MCT oil. An average increase (9.6%) of silicone compliance was measured along with oil migration in the tubing. Lipid uptake contributes significantly to deterioration of the silicone tested, leading to device failure. Surface damage can create a potential nidus for microorganisms, particularly fungi. Lipid type and rate of administration should be taken into account when long-term enteral feedings are given. These data contribute to the understanding of the causes of the physico-chemical deterioration of long-term enteral feeding devices and provide helpful information for the design and manufacture of improved products.

Title: Separation and estimation of

seven vasodilators using packed column supercritical fluid chromatography.

Abstract: This paper reports a method for an isocratic separation and simultaneous estimation of seven vasodilators: isosorbide mononitrate (ISMN), isosorbide dinitrate (ISDN), cyclandelate, nimodipine, amlodipine, pentifylline and pentoxifylline using packed column supercritical fluid chromatography

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Authors: Bhoir IC, Raman B, Sundaresan M, Bhagwat AM


Address: K.J. Somaiya College of Science, Chemistry Department, Vidya Vihar, Mumbai, India.

(SFC). An arbitrary choice of vasodilatory compounds with respect to their chemical structures was made to examine the viability of this technique for analysis of drugs and pharmaceuticals. Elution was performed on a RP-C18 column. SFC offers several degrees of freedom: temperature, pressure and modifier concentration to attain optimum resolution and sensitivity. The effects of these parameters on retention time have been studied using methanol modified carbon dioxide. The analytes were identified and measured by UV-detection. The chromatographic points of merit have been listed. Detection limits appear to be similar to those found in liquid chromatography. Modifier concentration does generally make major changes in retention and selectivity. A full scale validation for the seven vasodilators has been attempted and the statistical quality evaluated.

Title: Penetration of household

insecticides through different types of textile fabrics.

Authors: Saleh MA, Kamel A, el-Demerdash A, Jones J

Journal: Chemosphere. 1998 36(7) 1543-1552

Address: Texas Southern University, Department of Chemistry, Houston 77004, USA.

Abstract: Six different types of fabrics were compared for their ability to protect against human exposure to three different commercial household aerosol insecticides. Fabrics used in this investigation were, 100% cotton, cotton-polyester thermal underwear, cotton-polyester blend (twill), 100% acrylic, 100% wool and artificial silk (rayon). The household insecticides were, Black Flag (Ant and Roach Killer), Raid (Ant and Roach Killer) and Hot Shot (Wasp and Hornet Killer) containing propoxur, permethrin/pyrethrins and chlorpyrifos/allethrins as their active ingredients respectively. A fluorescent tracer, 4-methyl-7-diethyl amino coumarin was mixed with the aerosol (or equivalent aliquot) and sprayed onto cloth squares fitted on Whatman paper patches. The percentage of penetration through the cloth was quantified by the intensity of the fluorescence spectrum of each patch extract and the amount of the tracer recovered was calculated. The extract was concentrated to 1/10th of the volume to measure the content of each of the insecticides by supercritical fluid chromatography (SFC) using electron capture (ECD) and diode array detectors. Scanning electron microscope (SEM) images of the fabrics showed the geometry of the yarn. The results obtained from the fluorescence spectra, SFC and SEM showed that cotton-polyester (twill), cotton, wool and cotton thermal underwear were the least penetrable materials for the aerosols. On the other hand, acrylic and artificial silk (rayon) were the most penetrable cloth types.

Title: Packed column supercritical fluid

chromatographic separation and Abstract: A reproducible and fast method has been developed for the assay of acetaminophen,

estimation of acetaminophen, diclofenac sodium and methocarbamol in pharmaceutical dosage forms.

Authors: Patil ST, Sundaresan M, Bhoir IC, Bhagwat AM

Journal: Talanta 1998 47 3–10

Address: Patel Research Centre For Chemistry and Biological Sciences, Mithibai College Building, Vile Parle (West), Mumbai 400 056, India

methocarbamol, and diclofenac sodium in bulk and drug forms using packed column supercritical fluid chromatography employing internal standard method. The analytes were resolved by elution with supercritical fluid carbon dioxide doped with 11.1% (v:v) methanol on a Shendon-Phenyl (250 4.6 mm) 5 mm column with detection monitored spectrophotometrically at 225 nm. The densities and polarities of the mobile phase were optimised from the effects of pressure, temperature and modifier concentration on chromatograhic figures like retention time (tR, min), retention factor (k%) etc. Modifier concentration proved to be the most effective means for changing both retention and selectivity. Calibration data and recovery of the drug from spiked concentrations were determined to assess the viability of the method. The supercritical fluid chromatography (SFC) method was directly compared to an HPLC assay, developed in the laboratory, of the same analytes. With respect to speed and use of organic solvents SFC was found to be superior, while in all other aspects the results were similar to HPLC. The method has been successfully used for the assay of two formulations containing a combination of (A) acetaminophen and methocarbamol and (B) acetaminophen and diclofenac sodium. There was no interference from excipients. The present work validates the recent proposition that supercritical fluid chromatography using CO2 and modifiers is a viable, faster alternative to reverse phase HPLC.

Title: Identification of milk fat

triacylglycerols by capillary supercritical fluid chromatography-atmospheric pressure chemical ionization mass spectrometry.

Authors: Laakso P, Manninen P

Journal: Lipids. 1997 32(12) 1285-1295

Address: University of Turku, Department of Biochemistry and Food Chemistry,

Abstract: Identification of milk fat triacylglycerols was accomplished by capillary supercritical fluid chromatography (SFC) combined with atmospheric pressure chemical ionization mass spectrometry [(APCI)MS]. Supercritical carbon dioxide was the carrier fluid in SFC. Ionization was achieved by introducing vapor of ammonia in methanol into the ionization chamber which resulted in the formation of abundant [M + 18]+ and [M - RCCO]+ ions of triaclyglycerols. These ions defined both the molecular weight and the fatty acid constituents of a triacylglycerol, respectively. SFC on a nonpolar stationary phase provided an efficient separation of triacylglycerols according to the combined number of carbon atoms in the acyl chains a molecule. In addition to the identification of the major chromatographic peaks representing molecules with 26-54 acyl carbons, minor peaks representing triacylglycerols with an odd number of acyl carbons were separated and identified. Furthermore, compositional information on partially separated isobaric triacylglycerols, which differed substantially in the chain length of the fatty acyl residues, was achieved within some of the

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Finland peaks. A new finding of the present study was the formation of abundant [M + 18]+ ions of saturated triacylglycerols in addition to diagnostic fragment ions,being of primary importance in structure elucidation. This extends the applicability of capillary SFC-(APCI)MS in the analysis of both saturated and unsaturated triacylglycerols.

Title: Extraction of artemisinin and

artemisinic acid from Artemisia annua L. using supercritical carbon dioxide.

Authors: Kohler M, Haerdi W, Christen P, Veuthey JL


Address: University of Geneva, Laboratory of Mineral, Analytical and Applied Chemistry, Switzerland.

Abstract: Artemisinin (an antimalaric compound) and its major precursor artemisinic acid, isolated as the active principles of the medicinal plant Artemisia annua L., were extracted by supercritical fluid extraction (SFE) and analyzed by supercritical fluid chromatography (SFC) using a capillary column, coupled with a flame ionization detector (FID). With optimized operating conditions, artemisinin and artemisinic acid were quantitatively extracted at a flow-rate of 2 ml min-1 in less than 20 min. The supercritical fluid was composed of carbon dioxide and 3% methanol with temperature and pressure fixed at 50 °C and 15 MPa, respectively. From the kinetic curves, it appears that the extraction of artemisinin is not limited by the diffusion of the analyte from the plant into the extraction fluid but rather by the elution process. These conditions avoided degradation of the analyte and gave clean extracts ready to be analyzed by SFC. The SFE-SFC-FID method was successfully applied to six samples of A. annua containing various concentrations of artemisinin and artemisinic acid. Results were compared with two conventional liquid solvent extraction processes.

Title: Interaction of human IgE with

soluble forms of IgE high affinity receptors.

Authors: Liu J, Ruppel J, Shire SJ

Journal: Pharm Res. 1997 14(10) 1388-1393

Address: Genentech, Inc., Department of Pharmaceutical Research & Development,

Abstract: Interaction of human IgE with its high affinity receptor (Fc epsilon RI) on mast cells and basophils is an important step for initiating IgE mediated immune responses. To characterize the IgE and Fc epsilon RI interaction, we investigated this interaction in terms of stoichiometry and binding affinity in solution. The binding of IgE and IgE Fc epsilon RI alpha chain, the extracellular portion of IgE high affinity receptor (sFc epsilon RI alpha) was compared with the binding of IgE and IgE immunoadhesin (Fc epsilon RI alpha-IgG). The interaction was characterized by analytical ultracentrifugation, size exclusion chromatography, light scattering and ELISA. We show that the sFc epsilon RI alpha is only able to bind to one IgE, while the immunoadhesin can bind to two IgE. The interaction between IgE and Fc epsilon RI is very strong. Both forms of soluble receptors have similar

South San Francisco, California 94080, USA

intrinsic binding affinity with IgE. Both soluble receptors (Fc epsilon RI alpha-IgG and sFc epsilon RI alpha) can block the binding of IgE to its high affinity receptors on cell surface. The Fc epsilon RI alpha-IgG is a better IgE binding protein than sFc epsilon RI alpha at physiological relevant conditions. A humanized anti-IgE monoclonal antibody, rhuMAb E25 that also can block the binding of IgE to its high affinity receptors appears to bind to IgE at slightly different regions or in a different manner as the soluble forms of IgE receptors.

Title: Determination of polycyclic

aromatic hydrocarbons in waters by use of supercritical fluid chromatography coupled on-line to solid-phase extraction with disks.

Authors: Bernal JL, Nozal MJ, Toribio L, Serna ML, Borrull F, Marce RM, Pocurull E



Abstract: The potential of supercritical fluid chromatography (SFC) coupled on-line to solid-phase extraction (SPE) with disks for determining sixteen different polycyclic aromatic hydrocarbons (PAHs) was assessed. A preliminary study of the chromatographic separation was conducted that led to the use of SPE coupled to SFC for improved detection limits. Disks of two different materials, i.e., C18 and polystyrene-divinylbenzene, were assayed in terms of the variables influencing the extraction step. C18 disks provided the best results, with detection limits ranging from 0.1 to 1.5 micrograms l-1. The ensuing method was applied to river and tap water with good repeatability and reproducibility, and no interference from the sample matrix.

Title: Separation of polar solutes by

packed column supercritical fluid chromatography.

Authors: Berger TA

Journal: J Chromatogr A. 1997 785

Abstract: The application of packed column supercritical fluid chromatography (SFC) for the separation of polar analytes is reviewed. A comparison is drawn between the separation of polar and non-polar analytes on open-tubular column SFC. The different column materials used in packed column SFC are considered. The need for modifiers and additives to the carbon dioxide eluent for packed column separations is discussion and the influence of the instrumentation on the development of the method

85

3-33 Address: Berger Instruments,

125 Sandy Drive. Newark. DE 19713, USA

is described. The application of packed column SFC to a wide range of structure types of acidic, neutral and basic analytes and of chiral analytes is reviewed and the effect of the conditions are compared.

Title: Comparison of liquid and

supercritical fluid chromatography for the separation of enantiomers on chiral stationary phases.

Authors: Williams KL, Sander LC, Wise SA


Address: National Institute of Standards and Technology, Chemical Science and Technology Laboratory, Gaithersburg, MD 20899-0001, USA

Abstract: Comparisons of liquid (LC) and supercritical fluid chromatography (SFC) were conducted using commercially available chiral stationary phases (CSPs) bearing three different types of chiral selectors. Chiral compounds of pharmaceutical and agricultural interest were used to probe advantages of limitations of SFC relative to LC for enantiomeric separations. Column equilibrium and parameter optimization were generally accomplished more rapidly in SFC than in LC. Although improved resolution was often observed in SFC, analysis times were not always lower in SFC than in LC. In some instances, SFC provided separation capabilities not readily accessible in LC.

Title: A preliminary study of the analysis

of cannabis by supercritical fluid chromatography with atmospheric pressure chemical ionisation mass spectroscopic detection.

Authors: Backstrom B, Cole MD, Carrott MJ, Jones DC, Davidson G, Coleman K

Journal: Sci Justice. 1997 37(2) 91-97

Address: University of Strathclyde,

Abstract: A rapid method is described for the analysis of Cannabis products by supercritical fluid chromatography (SFC) coupled to atmospheric pressure chemical ionization-mass spectroscopic (APCI-MS) detection. The method had a shorter analysis time than GC-MS methods, without the need for derivatization prior to analysis. It was also faster than HPLC methods, with better resolution and definitive identification. Linearity of detector response to cannabidiol, delta 8-tetrahydrocannabinol, delta 9-tetrahydrocannabinol and cannabinol was established, the detection limits for mass on column being 0.55 ng, 1.20 ng, 0.69 ng and 2.10 ng respectively. The technique offers a means by which Cannabis products can be definitively identified in a single chromatographic run. Application to casework samples is described.

Department of Pure and Applied Chemistry, Glasgow, United Kingdom.

Title: On-line solid-phase extraction

coupled to supercritical fluid chromatography to determine phenol and nitrophenols in water.

Authors: Pocurull E, Marce RM, Borrull F, Bernal JL, Toribio L, Serna ML


Address: Universitat Rovira i Virgili, Department de Quimica, Tarragona, Spain.

Abstract: Determining of phenol and nitrophenols using solid-phase extraction on-line coupled to supercritical fluid chromatography (SFC) is studied. SFC quickly separated the compounds studied, in less than 6 min, and solid-phase extraction was used to decrease the limits of detection. C18, PLRP-S and a highly cross-linked styrene-divinylbenzene copolymer in a 10 x 3 mm I.D. laboratory-packed precolumn were tested comparatively as sorbents in the preconcentration step. Tetrabutylammonium bromide was used as ion-pair reagent in the extraction process to increase breakthrough volumes, mainly for phenol. Performance of the method was checked with tap and river waters.

Title: Postparturition changes in the

triacylglycerols of cow colostrum. Authors: Laakso P, Manninen P, Makinen J,

Kallio H Journal: Lipids. 1996

31(9) 937-943

Address: University of Turku, Department of Biochemistry and Food Chemistry, Finland

Abstract: The changes in the triacylglycerol (TAG) composition of colostrum fat of three cows were studied. In addition to the determination of fatty acid composition by gas chromatography, the distribution of TAG according to the acyl carbon number (ACN) and molecular weight was analyzed utilizing both supercritical fluid chromatography (SFC) and ammonia negative-ion chemical ionization mass spectrometry (MS). Colostrum TG contained substantially less stearic and oleic acids and more myristic and palmitic acids than the normal Finnish milk fat. The major trends in the changes of fatty acids and TAG were similar for each cow, although clear differences between individuals were found. During the first week of parturition, the proportions of short-chain fatty acids (C4-C10) typically increased as well as those of stearic and oleic acids, whereas the relative amounts of C12-C16 acids decreased, especially those of myristic and palmitic acids. Distinct changes occurred also in TAG distributions: the proportions of molecules with ACN 38-40 increased and those with ACN 44-48 decreased. Although there were distinct differences between individuals shortly after

87

delivery, both the fatty acid compositions and TAG distributions of the milk samples of the cows started to resemble each other after one week. The theoretical profiles of colostrum TAG calculated based on the fatty acid compositions differed clearly from the ACN distributions analyzed by SFC and MS. Thus, the analysis of TAG is essential, because the changes in molecular species composition of colostrum TAG cannot be estimated according to the fatty acid analysis alone.


in drug analysis: a literature survey.

Authors: Salvador A, Jaime MA, Becerra G, Guardia Mde L

Journal: Anal Bioanal Chem. 1996 356(2) 109-122

Address: University of Valencia, Department of Analytical Chemistry, Faculty of Chemistry, Doctor Moliner 50, E-46100, Burjassot, Spain.

Abstract: The applications of supercritical fluid chromatography to the analysis of drugs have been carefully revised from the literature compiled in the Analytical Abstracts until March 1994. Easy-to-read tables provide useful information about the state-of-the-art and possibilities offered by SFC in pharmaceutical analysis. The tables comprise extensive data about samples analyzed, pharmaceutical principles determined, solvents used and sample quantity injected, supercritical fluids and modifiers employed, injection system, instrumentation, experimental conditions for chromatographic separations (density, pressure, flow, temperature), characteristics of columns employed (type, support, length, diameter, particle film thickness, stationary phase), detectors, type of restrictors, and also some analytical features of the methods developed (such as retention time, resolution, sensitivity, limit of detection and relative standard deviation).

Title: Informational orthogonality of two-

dimensional chromatographic separations.

Authors: Slonecker PJ, Li X, Ridgway TH, Dorsey JG


Address: Henkel Corporation Emery Group, Technical Center, Cincinnati, Ohio 45232-1491, USA.

Abstract: A qualitative informational similarity technique has been used to describe the informational orthogonality of projected two-dimensional (2-D) chromatographic separations of complex mixtures from their one-dimensional 1-D separations. The reversed-phase liquid chromatography (RPLC), supercritical fluid chromatography (SFC), gas-liquid chromatography (GLC), and micellar electrokinetic capillary chromatography (MECC) retention behavior of up to 46 solutes of varying molecular properties was studied by 2-D range-scaled retention time plots and information entropy calculations. One hundred five combinations of technique/stationary phase pairs were used to simulate the 2-D chromatographic analyses. The informational entropy of one and two dimensions, the mutual information, the synentropy or "cross information", and the informational similarity were

calculated to describe the informational orthogonality. In addition, pattern descriptors were used to qualitatively describe the 2-D peak distribution. With the solutes tested, informational orthogonality, zero informational similarity, was observed with MECC-SDS/SFC-C1, MECC-SDS/SFC-Carbowax, MECC-TTAB/SFC-Carbowax, HPLC-C18/GLC-DB-5, HPLC-PBD/SFC-phenyl, SFC-Carbowax/GLC-DB5, and HPLC-phenyl/SFC-phenyl 2-D chromatographic systems. Conversely, with the solutes tested, informational nonorthogonal behavior described by range-scaled retention time plots to moderate to severe band overlap and data clustering was observed with 2-D chromatographic systems with high informational similarity and moderate to high degrees of synentropy. These results should prove useful for predicting complementary 2-D techniques as well as for choosing a second separation technique for confirmation of separation or peak purity.

Title: Plasma mass spectrometry as a

detector for chemical speciation studies.

Authors: Tomlinson MJ, Lin L, Caruso JA

Journal: Analyst. 1995 120(3) 583-589

Address: University of Cincinnati, Department of Chemistry, OH 45221-0172, USA

Abstract: Inductively coupled plasma mass spectrometry (ICP-MS), when coupled with the unique separating power of various chromatographic techniques, allows the detection of various elements at ultra-trace levels. The investigation of various toxic elements of environmental concern coupling relatively recent techniques, such as supercritical fluid chromatography (SFC) and capillary zone electrophoresis (CZE), to ICP-MS, is discussed. Comparisons have been made with detection limits obtained by using the flame ionization detector. The conventional technique of liquid chromatography for the speciation of vanadium, chromium and nickel is also discussed.

Title: A supercritical fluid

chromatographic method using packed columns for phenylbutazone and oxyphenbutazone in serum, and for phenylbutazone in a dosage form.

Abstract: The separation of phenylbutazone (PB) and its major metabolite oxyphenbutazone (OPB) using supercritical fluid chromatography (SFC) has been investigated. The separations were studied on octadecylsilane, silica and cyano packed columns with 5% methanol in carbon dioxide as mobile phase and detection at 240 nm. The octadecylsilane column showed the most favourable chromatographic parameters for the analysis of the analytes. Recoveries of PB and OPB from spiked human serum were in the 82-83% range using solid phase extraction on an ODS cartridge. Limits

89

Authors: Simmons BR , Jagota NK, Stewart JT


Address: University of Georgia, Department of Medicinal Chemistry, College of Pharmacy, Athens 30602-2352, USA.

of detection of the SFC assay were 0.1 microgram ml-1 for PB and 1.0 microgram ml-1 for OPB. Accuracy and precision of the method were in the 0.24-4.94% range for PB and OPB. The SFC method was directly comparable to an HPLC assay of the same analytes. The SFC method was also applied to a commercial 100 mg dosage form of PB with good recovery of PB.


chromatography of beta-blockers. Authors: Bailey CJ, Ruane RJ, Wilson ID


Address: Zeneca Pharmaceuticals, Department of Safety of Medicines, Mereside, Alderley Park, Macclesfield, Cheshire UK.

Abstract: Packed-column subcritical and supercritical fluid chromatography (SFC) of a total of 10 beta-blocking drugs was investigated on four stationary phases using CO(2)-methanol based eluents. The stationary phases studied were all bonded silicas and included Diol, aminopropyl, cyanopropyl, and "C1". The SFC of some of these compounds was possible with simple CO(2)-methanol based mobile phases, but in general, it proved to be necessary to add triethylamine as a competing base in order to obtain acceptable peak shapes. The best chromatographic results were obtained on the aminopropyl bonded phase, with good results also seen on the Diol phase. Different selectivities were observed for the Diol and aminopropyl columns.

Title: Analysis of felodipine by packed

column supercritical fluid chromatography with electron capture and ultraviolet absorbance detection.

Authors: Strode JT 3rd, Taylor LT, Howard AL, Ip D, Brooks MA


Address: Virginia Polytechnic Institute,

Abstract: A reproducible and selective supercritical fluid chromatography (SFC) method was developed for the analysis of felodipine, a drug indicated for the treatment of hypertension. Methanol-modified carbon dioxide was employed as the SFC mobile phase with both electron capture detection (ECD) and multi-wavelength detection (MWD) being used simultaneously for analyte determination. Chromatography limit of detection (LOD) and limit of quantitation (LOQ), linear dynamic range (LDR) and injection precision were obtained in order to assess chromatographic and detector performance for both the SFC/MWD and SFC/ECD/MWD systems. The method was shown to be stability indicating since felodipine could be separated from its potential oxidative degradation product, H152/37, in under 6 min (felodipine k' = 2.44). Sample throughput was increased by

Department of Chemistry, Blacksburg, VA 24061.

60% with the SFC assay vs LC. The optimized SFC method was shown to be equivalent to an existing LC/UV procedure for the analysis of a sustained-release tablet while realizing a 92% saving in disposable solvent waste. In order to achieve further solvent savings overall, supercritical fluid extraction (SFE) with 8% methanol-modified carbon dioxide as the extraction fluid was used to extract felodipine from a sustained-release tablet (as opposed to traditional solvent extraction). Comparable drug recoveries were obtained with SFE sample preparation technique when either SFC or LC extract analysis was utilized.

Title: Analysis of cholesterol and

cholesteryl esters in human serum using capillary supercritical fluid chromatography.

Authors: Kim DH, Lee KJ, Heo GS

Journal: J Chromatogr B Biomed Appl. 1994 655(1) 1-8

Address: Korea Research Institute of Standards and Science Organic Analytical Laboratory,

Abstract: Capillary supercritical fluid chromatography (SFC) using carbon dioxide as a mobile phase was applied for the determination of free cholesterol and cholesteryl esters in human serum. Serum samples were extracted with methanol-chloroform (2:1, v/v), and the extracts were analyzed by pressure programmed capillary SFC-flame-ionization detection (FID) without thermal degradation and derivatization. The total cholesterol concentrations obtained from SFC analysis were compared with those from GC or enzymatic analysis. The capillary SFC-FID method having high resolution gave an acceptable average relative standard deviation of 2.6%, and a detection limit of 4-6 pg. The quantitative results were acceptable for the simultaneous analysis of cholesterol and its esters in biological fluids. The concentration profiles of each compound in various samples, normal Korean human serum, Western human serum, and from high-cholesterol patient plasma, have been compared with this method.

Title: High-level expression of the

truncated alpha chain of human high-affinity receptor for IgE as a soluble form by baculovirus-infected insect cells. Biochemical characterization of the recombinant product.

Authors: Yagi S, Yanagida M, Tanida I,

Abstract: The binding subunit of human high-affinity receptor for IgE (Fc epsilon RI alpha) was efficiently expressed as a truncated form in insect cells. The soluble (s)Fc epsilon RI alpha purified from culture medium by affinity chromatography with an anti-(alpha chain) mAb was nearly homogeneous and had an IgE-binding activity. The amino acid composition and the revealed N-terminal amino acid sequence of sFc epsilon RI alpha suggested that it was properly processed in insect cells. The apparent molecular mass (35 kDa) of purified sFc epsilon RI alpha was smaller than that of sFc epsilon RI alpha produced by CHO transfectants. The reduction of the apparent

91

Hasegawa A, Okumura K, Ra C Journal: Eur J Biochem. 1994

220(2) 593-598

Address: Tonen Corporation, Corporate Research and Development Laboratory, Iruma-gun, Japan.

molecular mass after N-glycanase treatment showed the recombinant product was N-glycosylated. Peptide mapping of native and deglycosylated sFc epsilon RI alpha indicated that three Asn residues (Asn21, Asn42 and Asn166) should be almost fully glycosylated, and that two Asn residues (Asn74 and Asn135) were partially glycosylated.

Title: Chiral separations by packed-

column super- and subcritical fluid chromatography.

Authors: Anton K, Eppinger J, Frederiksen L, Francotte E, Berger TA, Wilson WH


Address: Ciba-Geigy Ltd., Pharma Research Department, CH-4002 Basle, Switzerland

Abstract: The higher diffirsivity and lower viscosity of supercritical and near-critical fluids can make packed-column supercritical fluid chromatography or subcritical fluid chromatography (SubFC) faster with improved resolution over normal-phase HPLC for chiral separations. However, superior fluid characteristics do not guarantee enhanced resolution or shorter analysis time. In the case of phenylalaninol, a ‘H NMR spectrum indicates interaction of the solute with carbon dioxide. Such interaction may explain the poorer resolution achieved by SubFC than by HPLC. For a secondary amine, its ‘H NMR spectra showed no shift while poorer resolution was again observed for SubFC when compared to HPLC. Thus, the NMR data by itself do not conclusively indicate either reaction between basic solutes and carbon dioxide or the likelihood of chiral resolution. The effects of column outlet pressure, organic modifier composition, pump flow-rate and column temperature for hydroxyzine were studied. Of the physical parameters studied, modifier composition has the greatest impact on retention. Increasing retention generally increases resolution. Changing temperature generally has less impact on retention but produces the greatest selectivity changes.

Title: Chromium determination by

supercritical fluid chromatography with inductively coupled plasma mass spectrometric and flame ionization detection.

Authors: Carey JM, Vela NP, Caruso JA

Abstract: Supercritical fluid chromatography (SFC) has been investigated for the separation of a pair of beta-ketonate chromium compounds and a thermally labile organochromium dimer. A limited comparison between flame ionization detection (FID) and inductively coupled plasma mass spectrometric (ICP-MS) detection of these compounds is presented. The beta-ketonate complexes were observed with both detectors, while the thermally labile dimer was not observed with ICP-MS


Address: University of Cincinnati, Department of Chemistry, OH 45221-0172.

detection. Detection limits for these compounds with ICP-MS were in the range of 0.9 to 3 pg with FID giving values between 10 and 250 pg. Reproducibility of the method is between 1 and 4% relative standard deviation (R.S.D.). The technique provided a linear response over approximately three orders of magnitude. The effect of two mobile phases (nitrous oxide and carbon dioxide) on the detection by each of the detectors are presented in a qualitative manner. Finally, the SFC-ICP interface heating method and the manner in which the restrictor is heated in the FID system are compared and there effect on the chromatography discussed.

Title: Soluble Fc gamma RIIIa is present

in plasma and is derived from natural killer cells.

Authors: de Haas M, Kleijer M, Minchinton RM, Roos D, von dem Borne AE

Journal: J Immunol. 1994 152(2) 900-907

Address: The Netherlands Red Cross Blood Transfusion Service, Central Laboratory, Amsterdam.

Abstract: Fc gamma RIII (CD16), a receptor for complexed IgG, is encoded by two very homologous genes: Fc gamma RIIIA and Fc gamma RIIIB. NK cells and macrophages express Fc gamma RIIIa, whereas only neutrophils constitutively express Fc gamma RIIIb. In a previous study we found that soluble (s)Fc gamma RIII in plasma seemed to originate only from neutrophils. However, CD16 mAb, directed against different epitopes of Fc gamma RIII, precipitated a glycoprotein from plasma of homozygous Fc gamma RIIIB gene-deficient donors. This glycoprotein migrated in a similar way as did released Fc gamma RIIIa derived from NK cells, whereas Fc gamma RIIIa released by cultured monocytes migrated differently and appeared to be more heavily glycosylated on SDS-PAGE. After deglycosylation, the M(r) of the plasma sFc gamma RIIIa was similar to that of released Fc gamma RIIIa. Moreover, V8-protease maps were identical. Therefore, we conclude that sFc gamma RIIIa is also present in plasma and is derived from NK cells. Because sFc gamma RIII levels are hardly detectable in the plasma of most homozygous Fc gamma RIIIB gene-deficient donors, we suspect that the sFc gamma RIIIa level is negligible compared with the level of sFc gamma RIIIb in plasma of healthy donors. Two patients with an NK cell lymphocytosis had a high plasma level of sFc gamma RIIIaNK. Furthermore, high levels of sFc gamma RIIIaNK were found in plasma of two patients with rheumatoid arthritis. Thus, the level of sFc gamma RIIIaNK might reflect either an increase in circulating NK cells or an enhanced release of Fc gamma RIIIaNK in certain diseases. This study shows that an assay that discriminates between sFc gamma RIIIa and sFc gamma RIIIb is necessary for the interpretation of sFc gamma RIII levels in patients.

93

Title: Advances in chromatography for clinical drug analysis: supercritical fluid chromatography, capillary electrophoresis, and selected high-performance liquid chromatography techniques.

Authors: Wong SH

Journal: Ther Drug Monit. 1993 15(6) 576-580

Address: Johns Hopkins University School of Medicine, Department of Pathology, Baltimore, Maryland.

Abstract: Advances have been made in chromatography complement immunoassay for clinical drug analysis. Chromatographic theory shows that the minimum detectable mass is directly proportional to the square of column radius. Small internal diameter columns are capable of analyzing lower analyte concentrations, and high resolution is achievable in open capillary columns, concomitant with greatly reduced mobile phase consumption and waste. This review, based on the author's experience and literature, focuses on supercritical fluid chromatography (SFC), capillary electrophoresis (CE), and selected high-performance liquid chromatography (HPLC) methodologies--microbore and direct-sample analysis (DSA) using commercially available Restricted Access Media (RAM) and REMEDi. In investigating the feasibility of SFC, the "normal-phase-like" selectivity of carbon dioxide was established, affecting the design of the extraction protocol and the elution order of drugs and metabolites. For example, the "more polar" tautomer eluted after FK-506, opposite to the order in the reversed-phase HPLC analysis. CE was investigated by Shihabi et al. for the analysis of pentobarbital and iohexol, while Evenson and Wiktorowicz performed preliminary evaluation of several therapeutic drug monitoring (TDM) drug groups. Innovation in HPLC column technology and hardware have greatly enhanced clinical drug analysis. Microbore column, offering enhanced mass sensitivity and high resolution, utilizes small sample size of 5 microliters of serum for the analysis of chloramphenicol. Commercially available RAM include internal surface reversed phase, shielded hydrophobic phase, and dual zone media, readily applicable for serum drug analysis without any sample preparation. Recently, an automated HPLC REMEDi offers urine and serum drug screening for toxicology.

Title: Chiral HPLC versus chiral SFC:

evaluation of long-term stability and selectivity of Chiralcel OD using various eluents.

Authors: Lynam KG, Nicolas EC


Abstract: The long-term stability of Chiralcel OD columns under varying HPLC and SFC conditions was evaluated. Two new columns from the same supplier lot were procured, and one was installed in an HPLC system, and the other was installed in an SFC system. Enantiomeric mixtures of trans-stilbene oxide and carbobenzyloxy phenylalaninol were repeatedly injected in both systems over several days. For HPLC, hexane was the primary solvent used, along with IPA, EtOH, MeOH/IPA, or EtOH/TFA modifier. Carbon dioxide was the primary SFC solvent, together with the similar modifiers

Address: Du Pont Merck Pharmaceutical Company, Deepwater, NJ 08023.

as the HPLC. Column performance was monitored by measuring resolution, theoretical plate number, and alpha between enantiomeric peaks. It was observed that, when the eluent strengths were adjusted to provide comparable retention times, the Chiralcel OD gave superior enantiomeric resolution when used in the SFC than the HPLC. Equilibration in between solvents was faster with SFC. The columns were demonstrated to be quite stable in both systems.

Title: Infusion of Fc gamma fragments

for treatment of children with acute immune thrombocytopenic purpura.

Authors: Debre M, Bonnet MC, Fridman WH, Carosella E, Philippe N, Reinert P, Vilmer E, Kaplan C, Teillaud JL, Griscelli C

Journal: Lancet. 1993 342(8877) 945-949

Address: Hopital Necker-Enfants Malades, Unite d'Immuno-Hematologie, Paris, France

Abstract: Treatment of acute immune thrombocytopenic purpura (ITP) with intravenous immunoglobulin (IVIG) induces partial or complete responses, shown by transient or persistent increases in platelet count. The clinical benefit could be due to blockade of the Fc gamma receptor (Fc gamma R); platelets sensitised by IgG could not be cleared by cells of the reticuloendothelial system if Fc gamma R on these cells was blocked with IVIG. To find out whether this putative mechanism is correct, we treated twelve children who had acute ITP with intravenous infusions of Fc gamma fragments. Eleven children showed rapid increases in platelet counts to above the critical value of 50 x 10(9)/L, thereby avoiding major haemorrhagic risk. The response was stable in six patients and transient in five. No adverse reactions were observed. In responders who had detectable platelet-associated IgG before treatment (> 1500 IgG per platelet), platelet IgG fell substantially with treatment. Serum soluble CD16 (sCD16 or sFc gamma RIII) concentrations, measured in five children, showed transient or stable increases that correlated with the rise in platelet count. No sCD16 was detected in the Fc gamma preparation used. We conclude that the infusion of Fc gamma fragments is an efficient treatment of acute ITP in children. The efficacy of Fc gamma fragments strengthens the hypothesis that Fc gamma R blockade is the main mechanism of action of IVIG in ITP, although other immunoregulatory mechanisms triggered by the presence of increased sCD16 concentrations in serum could be involved in the clinical benefit observed.

Title: Micro liquid chromatography-

mass spectrometry with direct liquid introduction used for separation and quantitation of all-

Abstract: The separation and quantitation of the pentafluorobenzyl derivatives of all-trans- and 13-cis-retinoic acids and their 4-oxo metabolites in human plasma on micro high-performance liquid chromatographic columns (0.32 mm I.D.) is described. The column outlet was directly coupled to

95

trans- and 13-cis-retinoic acids and their 4-oxo metabolites in human plasma.

Authors: Ranalder UB, Lausecker BB, Huselton C

Journal: J Chromatogr. 1993 617(1) 129-135

Address: Hoffmann-La Roche AG, Department of New Technologies, Basel, Switzerland.

the source of a quadrupole mass spectrometer via a simple SFC-frit interface. Negative ion chemical ionization conditions were obtained by coaxial introduction of ammonia as a reagent gas. A signal-to-noise ratio well above 3 was obtained for 1 pg of each analyte injected. The limit of quantitation calculated from spiked biological plasma extracts was 0.3 ng/ml.

Title: Rapid isolation of polychlorinated

biphenyls from milk by a combination of supercritical-fluid extraction and supercritical-fluid chromatography.

Authors: Mills AG, Jefferies TM

Journal: J Chromatogr. 1993 643(1-2) 409-418

Address: University of Bath, School of Pharmacy & Pharmacology, Claverton Down, UK.

Abstract: The extraction and isolation of polychlorinated biphenyls (PCBs) from sample matrices such as freeze-dried milk is a lengthy and expensive process that conventionally requires the isolation of microgram amounts of PCBs from large volumes of flammable organic solvents used in the extraction and chromatographic stages. Supercritical-fluid extraction (SFE) and chromatography (SFC) using carbon dioxide has been investigated as a replacement procedure using a Simplex optimisation approach to optimise the working conditions. The SFE conditions required to extract PCBs and milk fat from freeze-dried milk were investigated, and it was found that although extraction was rapid (10 min), the conditions required were identical so that there was no separation of PCBs and fat. However, SFC conditions have been developed that permit the resolution of PCBs from milk fat in 15 min. Thus, a combination of SFE and SFC have the potential to replace the existing methods of extraction and isolation with procedures that are quicker, cheaper, and avoid the use of flammable organic solvents.

Title: Comparison of flame ionization

and inductively coupled plasma mass spectrometry for the detection of organometallics

Abstract: Organotin compounds are separated by capillary supercritical fluid chromatography (SFC) and a comparison of the detection by flame ionization (FID) and inductively coupled plasma mass spectrometry (ICP-MS) is presented. Resolution, detection limits, linear dynamic range and

separated by capillary supercritical fluid chromatography.

Authors: Vela NP, Caruso JA


Address: University of Cincinnati, Department of Chemistry, OH 45221-0172.

reproducibility are the parameters compared between SFC-FID and SFC-ICP-MS, for the detection of tri- and tetraorganotin compounds. The resolution obtained in the SFC-FID system is not always observed in SFC-ICP-MS. Degradation in resolution is due to fluctuations in transfer line temperature. Baseline resolution for the organotins considered is achieved in both systems by using a longer column. Detection limits (DLs) are calculated as 3 sigma/S, where sigma is the standard deviation of the blank signal and S is the slope of the calibration curve. Detection limits of 10.3, 12.5, 12.0 and 9.0 pg are obtained for tetrabutyltin, tributyltin chloride, triphenyltin chloride and tetraphenyltin, respectively, using SFC-FID. An improvement in detection limits of one order of magnitude is achieved by SFC-ICP-MS for the same organotins (0.26, 0.80, 0.57 and 0.20 pg, respectively). The relative standard deviations using SFC-FID for five 50-nl injections, containing 0.5 ng Sn, ranged from 3.2 to 6.4%. Using SFC-ICP-MS, five replicate injections of 0.05 ng Sn give R.S.D.s from 1.3 to 3.4%.

Title: Interaction of the terminal

complement components C5b-9 with synovial fibroblasts: binding to the membrane surface leads to increased levels in collagenase-specific mRNA.

Authors: Jahn B, Von Kempis J, Kramer KL, Filsinger S, Hansch GM

Journal: Immunology. 1993 78(2) 329-334

Address: Universitat Heidelberg, Institut fur Immunologie, Germany.

Abstract: The late complement components, apart from their lytic function, are known to trigger the release of various proinflammatory substances from different types of nucleated cells. In the present study, the interaction of C5b-9 with synovial fibroblast cells (SFC) was examined. It was found that incubation of SFC with activated complement components resulted in binding of C5b-9 to the cell membrane; subsequently an increase in abundance of collagenase-specific mRNA was seen, as assessed by Northern blotting. When C8-deficient serum was used as source of complement neither binding of C5b-9 nor an increase in collagenase-specific mRNA could be detected. These findings suggest that C5b-9, which might be generated during rheumatoid inflammation, may contribute to chronic joint destruction by triggering collagenolytic activity.

Title: Recent advances in analytical

methods for mycotoxins. Abstract: Recent advances in analytical methods are reviewed using the examples of aflatoxins and

97

Authors: Gilbert J

Journal: Food Addit Contam. 1993 10(1) 37-48

Address: Ministry of Agriculture, Fisheries and Food, Food Science Laboratory, Norwich Research Park, Colney, UK

trichothecene mycotoxins. The most dramatic advances are seen as being those based on immunological principles utilized for aflatoxins to produce simple screening methods and for rapid specific clean-up. The possibilities of automation using immunoaffinity columns is described. In contrast for the trichothecenes immunological methods have not had the same general impact. Post-column derivatization using bromine or iodine to enhance fluorescence for HPLC detection of aflatoxins has become widely employed and there are similar possibilities for improved HPLC detection for trichothecenes using electrochemical or trichothecene-specific post-column reactions. There have been improvements in the use of more rapid and specific clean-up methods for trichothecenes, whilst HPLC and GC remain equally favoured for the end-determination. More sophisticated instrumental techniques such as mass spectrometry (LC/MS, MS/MS) and supercritical fluid chromatography (SFC/MS) have been demonstrated to have potential for application to mycotoxin analysis, but have not as yet made much general impact.

Title: Analysis of mono-, di- and

triglycerides in pharmaceutical excipients by capillary supercritical fluid chromatography.

Authors: Giron D, Link R, Bouissel S


Address: Sandoz Pharma Ltd, Analytical R+D, Basel, Switzerland.

Abstract: Mono-, di- and triglycerides are important components of oils, fats and other natural products. Since in general fatty acids are mixtures and glycerol can be differently substituted, finger-prints of the composition are suitable for better characterization. Since capillary supercritical fluid chromatography (SFC) employing carbon dioxide as mobile phase is compatible with flame ionization detection, it is possible to analyse many solutes at trace levels. Supercritical carbon dioxide offers higher solute diffusivity compared with the inert carrier gas conventionally used in gas chromatography and has a lower viscosity than the liquid solvents used in HPLC. Thus, glycerides of fatty acids can be separated and eluted at a lower temperature and with shorter analysis time in SFC. In this study the analysis of mono-, di- and triglyceride mixtures in several pharmaceutical excipients is reported using capillary SFC. Quantitative analysis is possible on the basis of a response factor established for each analyte. The accuracy of the method and its advantages are demonstrated.


of selected oestrogens. Abstract: The separation of selected oestrogens (oestrone, equilin, alpha-oestradiol, beta-oestradiol and d-

Authors: Jagota NK, Stewart JT


Address: University of Georgia, Department of Medicinal Chemistry, College of Pharmacy, Athens 30602.

equilenin) using capillary supercritical fluid chromatography (SFC) was studied. Three different stationary phases (SB-methyl-100, SB-biphenyl-30 and SB-cyanopropyl-50) were studied for the separation of the compounds. A baseline separation of the oestrogens was achieved on a SB-cyanopropyl-50 column using a carbon dioxide density gradient at an oven temperature of 73 degrees C. Typical analysis time on a 7 m column was 21 min. Retention times of each oestrogen decreased with an increase in either mobile phase density or oven temperature. Accuracy and precision of the SFC method were in the 1-5.5% range. The SFC method was applied to three different dosage forms containing oestrogens.

Title: Induction and function of Fc

epsilon RII on YT cells; possible role of ADF/thioredoxin in Fc epsilon RII expression.

Authors: Sorachi K, Sugie K, Maekawa N, Takami M, Kawabe T, Kumagai S, Imura H, Yodoi J

Journal: Immunobiology. 1992 185(2-4) 193-206

Address: Kyoto University Second Division of Internal Medicine, Faculty of Medicine, Medical School, Japan.

Abstract: The regulation of low-affinity Fc receptor for IgE (Fc epsilon RII) and the characteristics of both membrane and soluble forms of Fc epsilon RII were studied using YT cell line. We found that YT cells, a human NK like cell line, expressed Fc epsilon RII after IL-1 stimulation. Cross-linking of Fc epsilon RII on IL-1-stimulated YT cells as well as the transfectant of Fc epsilon RII-cDNA (YTSER) resulted in the up-regulation of IL-2R alpha (p55/Tac). A 59 kDa protein phosphorylated at tyrosine residues was co-immunoprecipitated with Fc epsilon RII from YTSER lysate using H107 anti-Fc epsilon RII mAb. YTSER not only expressed Fc epsilon RII on their surface but also secreted soluble form of Fc epsilon RII (sFc epsilon RII/sCD23; IgE binding factor). Affinity purification revealed that sFc epsilon RII released from YTSER is heterogeneous and consisted of several proteins differing in molecular weight. Both EBV+ B cells and HTLV-1+ T cells are high producers of ATL derived factor (ADF)/thioredoxin (TRX) and express Fc epsilon RII and IL-2R alpha respectively. To clarify the mechanism of Fc epsilon RII and IL-2R alpha induction by ADF/TRX, we examined the effect of ADF/TRX on the bindability of nuclear factor kappa B (NF-kappa B), which is known to regulate IL-2R alpha gene expression. In the gel shift assay, ADF/TRX was shown to enhance the bindability of NF-kappa B to its responsive element.

Title: Comparison of the carbohydrate

moieties of recombinant soluble Fc epsilon receptor (sFc epsilon

Abstract: Recombinant human soluble low affinity receptor for the Fc portion of IgE (sFc epsilon RII/sCD23) was produced in Saccharomyces cerevisiae or Chinese hamster ovary cells and subjected to

99

RII/sCD23) expressed in Saccharomyces cerevisiae and Chinese hamster ovary cells. Different O-glycosylation sites are used by yeast and mammalian cells.

Authors: Kalsner I, Schneider FJ, Geyer R, Ahorn H, Maurer-Fogy I

Journal: Glycoconj J. 1992 9(4) 209-216

Address: Bender & Co Ges mbH, Ernst Boehringer Institut fur Arzneimittelforschung, Vienna, Austria.

carbohydrate analysis. Applied methods included analytical SDS-PAGE, reversed phase HPLC, methylation analysis and sequential degradation with exoglycosidases. The results revealed that sFc epsilon RII derived from Chinese hamster ovary cells is glycosylated exclusively at Ser-147, containing mainly the trisaccharide Sia(alpha 2-3)Gal(beta 1-3)GalNAc, whereas the yeast derived glycoprotein was glycosylated at Ser-167 and contained only alpha-mannosyl residues. It is shown here for the first time that different amino acids of a given protein can be O-glycosylated when expressed in yeast or Chinese hamster ovary cells.

Title: Correlation between column

surface area and retention of polar solutes in packed-column supercritical fluid chromatography

Authors: Berger TA, Deye JF

Journal: J Chromatogr. 1992 594 2991-295

Address: Hewlett-Packard Co. P.O. Box 900, Avondale, PA 19311-0900, USA

Abstract: The retention of organic acids, amines, aminophenols, and amides was directly proportional to the surface area of Diol coated silica particles. Surface area was measured in situ using the method of Hong and Parcher [Anal. Chem., 62 (1990) 2313-23171. Linear regression analysis was performed on the results from four pore diameters (100, 300, 500 and 4000), plus the origin. Correlation coefficients between retention and surface area were as high as 0.9991. Both the surface area of the chromatographic support and solute retention changed by more than 14 times. All the solutes tested behaved similarly, indicating that changing the surface area is a viable means of retention control. The smallest pore diameter tested (100 A) produced the same efficiency as some of the larger pore sizes (i.e., 300 and 500 A) suggesting that still smaller sizes, like 60 A, might be useful for supercritical fluid chromatography. Particles with 4000 8, pores produced poor efficiency, due to peak tailing, which suggests that deactivation was inadequate.

Title: Separation of non-steroidal anti- Abstract:

inflammatory agents using supercritical fluid chromatography.

Authors: Jagota NK, Stewart JT


Address: University of Georgia, Department of Medicinal Chemistry, College of Pharmacy, Athens 30602.

Supercritical fluid chromatography (SFC) was investigated for the separation of non-steroidal anti-inflammatory agents (NSAIs). Three different stationary phases (SB-methyl-100, SB-biphenyl-30, and SB-cyanopropyl-50) were compared for the separation of the compounds. Baseline separation of a flufenamic acid, mefenamic acid, fenbufen and indomethacin mixture was achieved on the SB-biphenyl-30 column using a pressure gradient. A mixture containing flufenamic acid, mefenamic acid, acetylsalicylic acid, ketoprofen and fenbufen and another mixture containing ibuprofen, fenoprofen, naproxen, ketoprofen and tolmetin were well separated on the SB-cyanopropyl-50 column using pressure gradients. Typical analysis time for a mixture of NSAIs on the biphenyl or cyanopropyl column was approximately 20-25 min. Application of the method using the biphenyl column to the determination of NSAIs present in selected commercial dosage forms was demonstrated.

Title: Purification and characterization

of biologically active human recombinant 37 kDa soluble CD23 (sFc epsilon RII) expressed in insect cells.

Authors: Graber P, Jansen K, Pochon S, Shields J, Aubonney N, Turcatti G, Bonnefoy JY

Journal: J Immunol Methods. 1992 149(2) 215-226

Address: Glaxo Institute for Molecular Biology S.A., Geneva, Switzerland

Abstract: Human recombinant soluble 37 kDa CD23 has been expressed in insect cells and secreted into the culture medium using the IL-2 leader sequence. The 37 kDa CD23 was purified 600-fold to homogeneity by monoclonal antibody affinity chromatography and gel filtration. The pure protein is monomeric, glycosylated, depleted of one N terminal amino acid and contains four disulphide bonds. It degrades into smaller fragments of 33, 29 and 25 kDa if purified in the absence of protease inhibitors. The same pattern of proteolytic fragments is observed when the pure preparation is incubated at room temperature for 3 weeks. Physical characterization of the 37 kDa CD23 by circular dichroism indicates that the protein contains mainly beta sheet and 20% of alpha helical structures. Specific binding of IgE to natural CD23 (low affinity IgE receptor) was inhibited by purified recombinant 37 kDa CD23. Moreover, purified recombinant 37kDa CD23 and interleukin-1 promoted the survival of germinal centre B cells.

Title: Simultaneous determination of

arsenic and antimony species in environmental samples using

Abstract: Simultaneous separation and quantitation of arsenic(III) and antimony(III) can be achieved by extraction with lithium bis(trifluoroethyl)dithiocarbamate followed by supercritical fluid

101

bis(trifluoroethyl)dithiocarbamate chelation and supercritical fluid chromatography.

Authors: Laintz KE, Shieh GM, Wai CM


Address: University of Idaho, Chemistry Department, Moscow 83843

chromatographic (SFC) analysis. Arsenic(V) and antimony(V) are extracted after reduction with potassium iodide and sodium thiosulfate. Detection limits of 7 pg As and 11 pg Sb are achieved using this extraction method and SFC. Application to natural water and biological sample analysis is discussed.

Title: Determination of panaxadiol and

panaxatriol in ginseng and its preparations by capillary supercritical fluid chromatography (SFC).

Authors: Li YH, Li XL, Hong L, Liu JY, Zhang MY

Journal: Biomed Chromatogr. 1992 6(2) 88-90

Address: Second Military Medical University, College of Pharmacy, Shanghai, P.R. China.

Abstract: Capillary supercritical fluid chromatographic (SFC) method has been developed for the determination of panaxadiol and panaxatriol in ginseng and its preparations. 0.1 g ginseng or an appropriate amount of its preparations was hydrolysed by 15% H2SO4 in an ethanol:water (1:1 v/v) solution for 4 h followed by 15% NaOH for 0.5 h. The mixture was extracted by cyclohexane. The cyclohexane extracts were purified by a partition column and concentrated by an adsorption column and then analysed by SFC. Methyltestosterone was used as the internal standard.

Title: Direct enantiomeric separation of

beta-blockers on ChyRoSine-A by supercritical fluid chromatography: supercritical carbon dioxide as transient in situ derivatizing agent.

Abstract: The direct enantiomeric separation of a series of beta-blockers has been carried out on two chiral stationary phases (CSPs) derived from 3,5-dinitrobenzoyl tyrosine: the commercially available ChyRoSine-A and a recent improved version of this CSP. Using supercritical fluid chromatography (SFC), facile separations are achieved (1.1 less than Rs less than 7) within short analysis times. The parameters affecting the enantioselectivity (temperature, pressure, mobile phase nature, solute

Authors: Siret L, Bargmann N, Tambute A, Caude M


Address: Ecole Supérieure Chimie Industrielles de Paris, Laboratoire de Chimie Analytique, France.

structure) have been investigated. The optimal mobile phase consists in a mixture of carbon dioxide-methanol-propylamine at 25 degrees C. The solute structure has a great influence on the enantioselectivity. For instance, both amine and hydroxyl protons are necessary for chiral discrimination to occur. Furthermore, the steroselectivity value is directly connected to the amine substituent steric bulkiness. Surprisingly, these solutes are poorly resolved using normal phase liquid chromatography (NPLC). Accordingly, the specific influence of carbon dioxide on the enantiomeric separation of 1,2-amino-alcohols have been investigated using various techniques such as nuclear magnetic resonance (NMR) or molecular modelisation. It has been shown that carbon dioxide acts as a complexing agent toward the amino-alcohol by setting up of a bridge with the hydroxyl and the amine protons of the solute. In that way, the resulting complex possesses lower acido-basic properties and a higher conformational rigidity, responsible for chiral discrimination.

Title: Determination of oleanolic acid in

Achyranthes bidentata BLume and its preparations by supercritical fluid chromatography (Article in Chinese)

Authors: Wu MH, Li XL, Wang M, Liu JY, Zhang MY

Journal: Yao Xue Xue Bao. 1992 27(9) 690-694


Abstract: Capillary SFC was used for the determination of oleanolic acid in Achyranthes bidentata BLume and its preparations Tianmawan, Jinguishenqiwan and Hechedazaowan. The amount of oleanolic acid in the root of Achyranthes bidentata BLume was 1.75-2.19% (mean +/- SD = 1.94 +/- 0.17%). The limit of detection was 9.605 x 10(-11) g. The method is highly sensitive, accurate, reproducible, simple, rapid and specific, so, it can be extensively used for the determination of other preparations and crude herbs.


in the routine stability control of antipruritic preparations.

Authors: Anton K, Bach M, Geiser A

Abstract: A recently developed system for supercritical fluid chromatography (SFC), based on independent flow and pressure control and suitable for packed and capillary columns, was tested on a routine level for the reliable, accurate and precise determination of active pharmaceutical substances in

103

Journal: J Chromatogr. 1991 553(1-2) 71-79

Address: Ciba-Geigy Ltd., Pharma Analytical Department, Basle, Switzerland.

stability control. Only packed columns were used for this analysis. The chromatographic figures of merit and the validation data of the active substance alone and in two different dosage forms (accuracy, 98.8-99.2%; precision, 0.6%; linearity of response, 0.998-0.999) are comparable with the former liquid chromatographic methods. Economical (reduction of analysis time, fewer experimental steps and less sample pre-separation) and ecological (carbon dioxide of organic solvents) advantages make SFC an attractive alternative to liquid chromatography in the determination of crotamiton.

Title: Evaluation of inductively coupled

plasma mass spectrometry as an elemental detector for supercritical fluid chromatography.

Authors: Shen WL, Vela NP, Sheppard BS, Caruso JA


Address: University of Cincinnati, Department of Chemistry, Ohio 45221-0172.

Abstract: Supercritical fluid chromatography coupled with inductively coupled plasma mass spectrometry shows high potential for the determination at ultratrace levels of organometallic compounds of environmental interest. In this study the determination of organotin compounds at ultratrace levels is demonstrated. In this work a supercritical fluid chromatography/inductively coupled plasma mass spectrometry (SFC/ICPMS) interface was developed. Separation of tetraalkyltin compounds shows detection levels in the subpicogram range (0.034 pg for tetrabutyltin; 0.047 pg for tetraphenyltin). The linear ranges are over 3 orders magnitude (1-1000 pg). The reproducibility of sample injections are better than 5% RSD.

Title: Characterization of N-linked

glycans by supercritical fluid chromatography-mass spectrometry.

Authors: Sheeley DM, Reinhold VN

Journal: Anal Biochem. 1991 193(2) 240-247

Abstract: N-Linked glycans have been characterized by supercritical fluid chromatography (SFC) and SFC-MS using positive- and negative-ion chemical ionization. Four common oligosaccharide derivatives have been prepared and their chromatographic properties assessed on three SFC columns of varying polarity. Carbon dioxide has been used as the SFC mobile phase, with ammonia or CO2 added to the ion source for positive- and negative-ion chemical ionization, respectively. Direct SFC-MS interfacing allows the analytical manipulations of single-ion monitoring, total-ion plots, background

Address: Harvard School of Public Health, Department of Nutrition, Boston, Massachusetts 02115.

subtraction, library searches, and spectral reconstruction algorithms. Positive ammonia chemical ionization yields abundant molecular-weight information, (MH)+, and (MNH4)+ with little or no fragmentation. To capitalize on sensitivity, samples were prepared with the pentafluorobenzyl aminobenzoate reagent, acetylated, and analyzed by SFC-NICI-MS. This modification improves column efficiency and resolution and greatly enhances detecting sensitivity. These "soft" ionization conditions provide abundant molecular-weight-related anions for collision-induced dissociation and subpicogram detection.

Title: Role of additives in packed

column supercritical fluid chromatography: suppression of solute ionization.

Authors: Berger TA, Deye JF


Address: Hewlett-Packard Co. P.O. Box 900, Avondale, PA 19311-0900, USA

Abstract: Tailing of polycarboxylic acids in packed column supercritical fluid chromatography (SFC) is primarily due to solute ionization. Very acidic additives improve the peak shape of acidic solutes by suppressing solute ionization. Coverage of active sites appears to be a secondary function of additives. The sorption of acidic additives was measured on five stationary phases used in packed column SFC. Surface coverages (at constant mobile phase composition) varied by more than 50-fold (0.4 to 21%). depending on the stationary phase identity. Within the group of additives used, coverage was independent of additive identity or concentration but was inversely proportional to modifier concentration. Chromatographic peak shapes of polyfunctional acidic solutes were also observed under the same conditions as used for the surface coverage measurements. Solute peak shapes depended on the acid strength of the additives but were unrelated to the amount of additive retained on the column.

Title: Analysis of sulphonamides using

supercritical fluid chromatography and supercritical fluid chromatography-mass spectrometry.

Authors: Perkins JR, Games DE, Startin JR, Gilbert J

Journal: J Chromatogr. 1991 540(1-2)

Abstract: Packed-column supercritical fluid chromatography has been used for the separation of mixtures of sulphonamides on silica and amino-bonded stationary phases utilizing carbon dioxide with methanol modifier as the mobile phase. The effect of modifier concentration, column pressure and modifier identity on retention was also studied. Packed-column supercritical fluid chromatography-mass spectrometry (SFC-MS) of these mixtures utilizing both moving-belt and modified thermospray interfaces was also studied. The identification of sulphamethazine in a spiked porcine kidney extract was performed by SFC-MS using the moving-belt interface.

105

239-256 Address: University College of Swansea,

Department of Chemistry, U.K.

Title: Characterization of

glycosphingolipids by supercritical fluid chromatography-mass spectrometry.

Authors: Merritt MV, Sheeley DM, Reinhold VN


Address: Harvard School of Public Health, Boston, Department of Nutrition, Massachusetts 02115

Abstract: Gangliosides have been characterized by supercritical fluid chromatography-chemical ionization mass spectrometry (SFC-CIMS) as permethyl and pertrimethylsilyl derivatives, using carbon dioxide as the SFC mobile phase and CI reagent gas. Ganglioside classes and ceramide heterogeneity within each class are well resolved by SFC. Direct SFC-interfacing allows the analytical manipulations of single-ion monitoring, total-ion plots, background subtraction, library searches, and spectral reconstruction algorithms. Addition of ammonia to the CI ion chamber (NH3 as a CI reagent gas) yields abundant molecular-weight-related ions, (MH)+ and (MNH4)+ from analyte derivatives. Substitution of methanol for ammonia yields considerable parent-ion fragmentation, providing structural information on carbohydrate sequence, fatty acid, and sphingoid components. Under these latter conditions a unique alpha-cleavage fragment is observed which differentiates fatty acid from sphingosine heterogeneity. For ganglioside samples, the carboxyl group of neuraminyl residue(s) have been esterified with pentafluorobenzyl bromide and the products analyzed by negative ion chemical ionization MS. This modification improves chemical selectivity and greatly enhances detecting sensitivity. These "soft" ionization conditions provide abundant molecular-weight-related anions for collision-induced dissociation and subpicogram detection.

Title: Analysis of prostaglandins in

aqueous solutions by supercritical fluid extraction and chromatography.

Authors: Koski IJ, Jansson BA, Markides KE, Lee ML

Journal: J Pharm Biomed Anal. 1991 9(4)

Abstract: Trace amounts of prostaglandins (PGs) were selectively analysed without derivatization using supercritical fluid extraction (SFE) and open tubular column supercritical fluid chromatography (SFC). The specific compounds studied were prostaglandin F2 alpha (PGF2 alpha), esters of PGF2 alpha, prostaglandin F1 alpha) and prostaglandin E2 (PGE2). An open tubular column was used with carbon dioxide as the mobile phase and with universal flame ionization detection. Samples were introduced into the column by direct injection using a 1-microliter sample loop or by SFE with solute focusing. The 11 standard compounds were effectively separated within 35 min using a

281-290 Address: Brigham Young University,

Department of Chemistry, Provo, UT 84602

density program at constant temperature. The minimum detectable quantity (signal-to-noise ratio = 3) using the direct injection method was 9 ng for 15-propionate PGF2 alpha isopropyl ester. Using the extraction method, the sample size in the extraction cell was increased to 100 microliters, which made it possible to analyse compounds that were present in low concentrations. Aqueous PG samples were extracted from adsorbents onto which the samples had been loaded.

Title: Determination of panaxadiol and

panaxatriol in radix notoginseng and Yunnan baiyao by capillary supercritical fluid chromatography (Article in Chinese)

Authors: Li YH, Li XL, Hong L, Liu JY, Zhang MY

Journal: Yao Xue Xue Bao. 1991 26(10) 764-767


Abstract: Capillary supercritical fluid chromatography (SFC) was developed for the determination of panaxadiol and panaxatriol in Radix notoginseng and Yunnan baiyao. 0.1g Radix notoginseng powder or 0.5g Yunnan baiyao was mixed with 10 ml 15% H2SO4 ethanol-water (1:1) solution, adding 1 mg cholesterol as internal standard. The mixture was refluxed for 4 h, then adding 15 ml 15% NaOH solution, refluxed for 0.5 h. The mixture was extracted 3 times with 10 ml portions of cyclohexane. The cyclohexane extracts were purified by partition column and concentrated by adsorption column and then analysed by SFC. The proposed method is sensitive, accurate, precise, simple and rapid; all the process can be done in 8 h.

Title: Composition and density effects

using methanol/carbon dioxide in packed column supercritical fluid chromatography.

Authors: Deye JF, Berger TA

Journal: Anal Chem.1990 62 1181-1185

Address: Hewlett-Packard, P.O. Box 900, Route 41 and Starr Road,

Abstract: The density of mixtures of carbon dloxlde with 0-1 1.5 mol % methanol was measured at 40 OC, between 80 and 200 bar. Chromatograms were run at constant den- but varying compodtlon and at constant composition but varying dendty. For the systems studied, changing fluid compodtlon provldes wider adlustment of solute retentlon than density changes. Plots d log k’vs percent modifier, at constant density, were nonlinear. However, plots of log k’vs mobile phase solvent strength, as meawed using the solvatochromic dye Nile Red, produced a linear relatknship. The change In retentbn vs the pore volume (proportlonal to surface area) was also measured.

107

Avondale, Pennsylvania 19311

Title: Nile red as a solvatochromic dye for measuring solvent strength in mormal liquids and mixtures of normal liquids with supercritical and near critical fluids.

Authors: Deye JF, Berger TA, Anderson AG

Journal: Anal Chem.1990 62 615-622

Address: Hewlett-Packard, P.O. Box 900, Route 41 and Starr Road, Avondale, Pennsylvania 19311

Abstract: Nlle Red was used as a solvatochromic probe to measure the solvent strength of 85 pure liquids and the results were compared to solvent strength scales from the better known Relchardt’s dyes. Nile Red is also soluble in some pure supercritical fluids and in polar mixtures of supercritical fluids and modifiers. The solvent strength of mixtures of organic modifiers in CO2, Freon-13, and Fr-23 was measured as a function of modifier concentration with Nile Red and other solvatochromic dyes. Nile Red exhibits large shifts in the wavelength of its absorption maximum allowing subtle changes in solvent strength with fluid composition to be quantified, and it is stable in very strong acids. Relative scales of solvent strength comparing different modifiers added to C02 are presented and compared to a wide range of normal liquids.

Title: Femtomole oligosaccharide

detection using a reducing-end derivative and chemical ionization mass spectrometry.

Authors: Caesar JP Jr, Sheeley DM, Reinhold VN


Address: Harvard University School of Public Health, Boston, Massachusetts 02115.

Abstract: A bimodal reagent (pentafluorobenzyl aminobenzoate) has been synthesized to improve oligosaccharide isolation, detection, and structural characterization. The reagent is glycosidically attached to the reducing end of glycan residues, imparts fluorescent and uv properties for chromatographic detection, and functions as an efficient electron trap under negative ion chemical ionization mass spectrometry for femtomole detectability. Facile ester cleavage and pentafluorobenzyl elimination provides a single molecular-weight-related fragment in high abundance. Procedures are described for reagent synthesis, purification, and oligosaccharide conjugation. Carbohydrate samples derivatized with this reagent are evaluated by high-performance liquid chromatography and supercritical fluid chromatography (SFC) and for sensitivity by SFC negative ion chemical ionization mass spectrometry.

Title: Comparison of high-performance

liquid chromatography, Abstract: High-performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC) and

supercritical fluid chromatography and capillary zone electrophoresis in drug analysis.

Authors: Steuer W, Grant I, Erni F


Address: Sandoz Ltd., Pharma Division, Basle, Switzerland.

capillary zone electrophoresis (CZE) are compared with respect to their usefulness in drug analysis. Factors discussed include efficiency, performance, sensitivity, optimization parameters, method development time, sample preparation, technical difficulties, orthogonality of the information obtained and the possible application to various substance groups. It is concluded that HPLC can be applied successfully in virtually all areas of pharmaceutical analysis. CZE has a promising future in the analysis of drugs and in the field of biotechnological analysis, where a high number of plates is required together with a short analysis time. Nevertheless improvements in detection are still necessary for most applications. SFC is particularly suitable for moderately polar compounds or substances for which mass-sensitive detection is required. SFC and CZE can be considered as complementary to HPLC owing to the orthogonality of the acquired data, and as a result more information can be obtained from the analysis.

Title: Determination of mefloquine in

blood by supercritical fluid chromatography with electron-capture detection.

Authors: Mount DL, Patchen LC, Churchill FC


Address: Control Technology Branch, Centers for Disease Control, Atlanta, GA 30333.

Abstract: Supercritical fluid chromatography (SFC) with electron-capture detection is described for the sensitive quantification of mefloquine in 0.1-ml blood samples. The method is internally standardized and incorporates partitioning into methyl tert.-butyl ether (MTBE) from aqueous base, back-extraction into dilute aqueous acid and final partitioning into MTBE from aqueous base. SFC conditions include a silica-gel-packed, glass-lined steel column and a mobile phase of 0.15% n-butylamine and 1% methanol in supercritical n-pentane. The method has a detection limit of 7.5 ng/ml in 0.1-ml blood samples and exhibits good linearity and precision. The method compares favorably with a published high-performance liquid chromatographic procedure in the analysis of blood from volunteers who received mefloquine hydrochloride (15 mg as base per kg body weight).

Title: Measurement of the humoral

immune response against Streptococcus pneumoniae type 14-derived antigens by an ELISA and ELISPOT assay based on biotin-avidin technology.

Abstract: A Streptococcus pneumoniae type 14-specific ELISA and ELISPOT assay have been developed based on the use of biotinylated type 14 capsular polysaccharide (S14PS-biotin). A major advantage of this application over other methods is the use of 10-100-fold less antigen than that reported in the literature for other similar assays. Moreover, the prepared biotinylated polysaccharides are very stable and it is possible to use the same procedures for other pneumococcal polysaccharide

109

Authors: Verheul AF, Versteeg AA, Westerdaal NA, Van Dam GJ, Jansze M, Snippe H

Journal: J Immunol Methods. 1990 126(1) 79-87

Address: Utrecht University, Medical School, Eijkman-Winkler Laboratory of Medical Microbiology, The Netherlands.

antigens (e.g., S6BPS) with no major changes necessary in the ELISA and ELISPOT protocols. Furthermore, a simple thin layer chromatography method has been developed as a method for quality control of the biotinylated polysaccharide. Immunization with the thymus-independent antigen S14PS resulted in the induction of IgM spot-forming cells (SFC) and antibodies while S14PS-protein conjugates induced a thymus-dependent response. The immune response to the conjugates was enhanced by the addition of the adjuvant Quil A resulting in high levels of both IgG SFC and antibodies at day 14 after immunization. The developed assays are reliable and reproducible tools for studying the humoral immune response against Streptococcus pneumoniae type 14 capsular polysaccharide derived antigens.

Title: High-performance liquid

chromatography and capillary supercritical-fluid chromatography separation of vegetable carotenoids and carotenoid isomers.

Authors: Schmitz HH, Artz WE, Poor CL, Dietz JM, Erdman JW Jr


Address: University of Illinois, Department of Food Science, Urbana 61801.

Abstract: Carotenoids from carrots and tomatoes were separated with high-performance liquid chromatography (HPLC) and capillary supercritical fluid chromatography (SFC). All trans alpha- and beta-carotene were separated from their respective cis-isomers with capillary SFC. Carotenoids extracted from tomatoes included xanthophyll, lycopene and beta-carotene, while alpha- and beta-carotene were extracted from carrots. The HPLC separations were accomplished isocratically with a 25-cm column containing 5-microns ODS and methanol-acetonitrile-chloroform (47:47:6) or acetonitrile-dichloromethane (80:20). beta-Carotene cis-isomers were separated with SFC with a SB-cyanopropyl-25-polymethylsiloxane column, while alpha-carotene isomers were separated with two SB-cyanopropyl-50-polymethylsiloxane columns. Carotenoids from carrots and tomatoes were separated with a SB-phenyl-50-polymethylsiloxane column. Carbon dioxide with 1% ethanol was the SFC mobile phase. The eluent was monitored at 461 nm for HPLC and either 453 or 461 nm for SFC.

Title: Effects of temperature and density

on retention in capillary supercritical fluid chromatography.

Authors: Berger TA

Abstract: Retention of fluoranthene using carbon dioxide and a methyl silicone capillary column was measured at multiple, constant densities while changing temperature. Log k’ VS. l/T plots (at constant density) yielded straight lines with different slopes. Each slope gave a value for the total enthalpy of interaction between solute, mobile, and stationary phase, at each density. Gas


Address: Hewlett-Packard P.O. Box 900, Route 41 and Starr Road, Avondale, PA 19311, U.S.A.

chromatographic measurements using the same column and low pressure carbon dioxide gave a value for the interaction of the solute and the stationary phase. The difference between the total enthalpy and the solute-stationary phase enthalpy gave an approximate value for the solute-mobile phase enthalpy of interaction. Solubility problems emerged as temperature was decreased while density was held constant.

Title: Bioanalytical applications of


Authors: Niessen WM, Tjaden UR, van der Greef J


Address: Center for Bio-Pharmaceutical Sciences, Division of Analytical Chemistry, RA Leiden, The Netherlands.

Abstract: Supercritical fluid chromatography (SFC) has become a valuable tool in analytical chemistry. In the past few years considerable progress has been made in bioanalytical applications of both capillary and packed column SFC. Progress in this respect has especially been made with combined SFC-mass spectrometry, an important alternative to combined liquid chromatography-mass spectrometry. The various applications of capillary and packed column SFC are reviewed, while special attention is given to detection technique applicable in (bioanalytical) SFC. Considerable progress in SFC is expected in the near future.

Title: Preparation and evaluation of a

99mTc-SnF2 colloid kit for leukocyte labeling.

Authors: Hirsch JI, Tatum JL, Fratkin MJ, Apostolides DL, Quint RI

Journal: J Nucl Med. 1989 30(7) 1257-1263

Address: Medical College of Virginia, Department of Radiology, Virginia

Abstract: Stannous fluoride colloid (SFC) kits for instant radiolabeling with 99mTc were prepared and evaluated for suitability as a leukocyte radiolabeling agent. Technetium-99m labeling for kits stored at -15 degrees C for up to 3 mo was greater than 95% as determined by instant thin layer chromatography while colloid particles of 1-3 microns were measured by electron microscope for these preparations. Canine leukocyte preparations labeled with [99mTc]SFC and characterized by triple density gradients of metrizamide in plasma demonstrated an 83% leukocyte association. Analysis of labeled cell preparation for up to 3 hr demonstrated label stability. Labeled leukocytes, when readministered in normal dogs, demonstrated bi-exponential blood clearance with uptake and subsequent clearance from lung. There was increasing uptake of labeled leukocytes by the liver until

111

Commonwealth University Health Sciences Division, Richmond.

steady state was achieved. Furthermore, when whole blood samples were analyzed by the triple density gradient method, an increasing monocyte-to-granulocyte ratio was observed to occur with time. By 3 hr 95% of the whole blood activity was associated with the leukocyte fraction. Dogs in which a 24-hr sterile abscess was created demonstrated elevated blood-pool activity as compared to control with localization of the labeled cells at inflammatory sites within 3 hr following cell readministration.


and microbore liquid chromatography for drug analysis.

Authors: Wong SH

Journal: Clin Chem. 1989 35(7) 1293-1298

Address: University of Connecticut School of Medicine, Department of Laboratory Medicine, Farmington 06032.

Abstract: Supercritical fluid and microbore liquid chromatography offer potential applications for drug analysis. In supercritical fluid chromatography (SFC), the mobile phase is a gas (e.g., carbon dioxide) maintained at its supercritical state--that is, above its critical temperature and pressure, above which it cannot be liquefied even with further increases in applied pressure. The SFC mobile phase has low viscosity, approximating that of a gas, and high diffusivity, between those of a gas and a liquid. These properties yield favorable column efficiency, between that of capillary gas chromatography (GC) and liquid chromatography (LC). SFC analysis may be performed by either packed or open tubular capillary columns and with GC and LC detectors. SFC, interfaced with mass spectrometry, may become a viable alternative to GC/MS for drug identification in clinical and forensic toxicology. Advantages of microbore liquid chromatography include enhanced mass sensitivity, reduced solvent consumption, and others. Microbore columns (internal diameters 1 to 2 mm) may be packed with 3-, 5-, or 10-micron particles. Potential applications include micro-sample analysis (5-200 microL) for neonatal and pediatric drug monitoring, and drug confirmation analysis for toxicology.


chromatography/mass spectrometry via a two-stage momentum separator.

Authors: Edlund PO, Henion JD

Journal: J Chromatogr Sci. 1989

Abstract: The practical utility of a two-stage momentum separator for combining packed-column supercritical fluid chromatography (SFC) with mass spectrometry (MS) is described. A Hewlett-Packard model 1084B liquid chromatograph modified for packed-column SFC is connected to a linear fused-silica capillary restrictor housed in a heated probe held at 60 degrees at the terminus. A makeup of coaxial helium gas (1.5 L/min) or dissolved solvent (0.2-0.4 mL/min) can be introduced at the

27(6) 274-282

Address: Cornell University, New York State College of Veterinary Medicine, Ithaca 14850.

point of supercritical fluid expansion. The latter SFC effluent (0.3-2.0 mL/min) is expanded into a heated (44 degrees) desolvation chamber and directed through a nozzle positioned at the entrance of a two-stage momentum separator. Enrichment of the analyte relative to the volatile gases allows the transfer of sample particles to the MS ion source to produce electron ionization of flash-volatilized eluates. On-line SFC/MS separation and detection of low microgram levels of involatile, thermally labile analytes in synthetic mixtures is accomplished. Identification of an unknown compound in a drug tampering incident and the identification of an unknown metabolite isolated from horse urine is also accomplished.

Title: Analysis of butter fat

triacylglycerols by supercritical fluid chromatography/electron impact mass spectrometry.

Authors: Kallio H, Laakso P, Huopalahti R, Linko RR, Oksman P


Address:

Abstract: Triacylglycerols (TAGs) from butter fat isolated by solvent extraction were analyzed by use of a capillary column supercritical fluid chromatograph (SFC) combined with a flame ionization detector or a double focusing mass spectrometer. The chromatographic separation was achieved by using a dimethyldiphenylpolysiloxane phase (DB-5) to bundle up the TAGs with the same carbon number. The ratio of TAGs with varying degree of unsaturation in each SFC peak was determined by using the selected ion monitoring of the molecular ions with electron impact mode. The discrimination between the fatty acids at the position sn-2 and positions sn-1/3 in a triacylglycerol molecule was demonstrated by monitoring the ions [M - RCO2CH2]+ from reference compounds.

Title: Linear velocity controm in

capillary supercritical fluid chromatography by restrictor temperature programming.

Authors: Berger TA, Toney C


Address: Hewlett-Packard P.O. Box 900,

Abstract: Increasing restrictor temperature decreases mass flow through capillary columns in a predictable way. Changes in mass flow can be related to changes in column linear velocity. Restrictor temperature programming, therefore, offers the potential for independent control of column efficiency while pump pressure, at constant oven temperature, controls fluid density. Experimental measurements of mass flow through restrictors at various temperatures and pressures confirm model predictions.

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Route 41 and Starr Road, Avondale, PA 19311, U.S.A.

Title: Enantiomer resolution of D- and L-

alpha-amino acid derivatives by supercritical fluid chromatography on novel chiral diamide phases with carbon dioxide.

Authors: Dobashi A, Dobashi Y, Ono T, Hara S, Saito M, Higashidate S, Yamauchi Y


Address: Tokyo College of Pharmacy, Japan.

Abstract: The rapid resolution of racemic N-4-nitrobenzoylamino acid isopropyl esters was accomplished without the loss of enantioselectivity by supercritical fluid chromatography (SFC) on novel chiral valine-diamide phases with carbon dioxide and a polar methanol modifier. In each stationary phase, a chiral moiety was anchored to the silica gel surface by a long decamethylene spacer. The enantioselectivity in SFC was comparable to that in liquid chromatography using 2-propanol-n-hexane. The time required for analysis was less than 5 min, and the range of enantiomer resolution (Rs) was 10.8-1.25. On using 2-propanol in place of methanol the separation was improved, but was accompanied by a decrease in column efficiency. The end-capping effect of the remaining surface silanols on enantiomer resolution is discussed.

Title: Analysis of steroids by capillary

supercritical fluid chromatography with phosphorus-selective detection.

Authors: David PA, Novotny M


Address: Indiana University, Department of Chemistry, Bloomington 47405

Abstract: Steroids were derivatized with dimethylthiophosphinic chloride to produce steroidal thiophosphinic esters. A catalyst, 4-dimethylaminopyridine, was used to promote quantitative and reproducible thiophosphinic ester formation at low reaction temperatures. The derivatives were analyzed by capillary supercritical fluid chromatography (SFC) with phosphorus thermionic detection. The phosphorus thermionic detector exhibited linearity over 3-4 orders of magnitude. A sensitivity of 120 fg P/s was obtained for the dithiophosphinic ester of pregnanediol at a signal-to-noise ratio of 3. The efficiency of capillary SFC and the excellent sensitivity of the phosphorus thermionic detector were demonstrated by the analysis of steroids isolated from both human urine and plasma.

Title: Fluid chromatography in

supercritical conditions: a method Abstract: The behaviour of high polar polyphenolic compounds (taxifolin and the flavolignans of the silymarin

potentially useful for flavonolignan analysis (Article in French).

Authors: Martinez F

Journal: Ann Pharm Fr. 1989 47(3) 162-168

Address:

complex) by supercritical fluid chromatography (SFC), using standard HPLC packed columns (0.46 cm i.d.) and CO2 as the primary mobile phase, was studied. The elution and separation of the flavonolignans by some of the systems investigated was shown

Title: Phase-system switching as an on-

line sample pretreatment in the bioanalysis of mitomycin C using supercritical fluid chromatography.

Authors: Niessen WM, Bergers PJ, Tjaden UR, van der Greef J


Address: Leiden University, Division of Analytical Chemistry, Gorlaeus Laboratories, The Netherlands.

Abstract: One of the problems of the application of supercritical fluid chromatography (SFC) in bioanalysis is the fact that many sample pretreatment procedures deliver the solutes of interest in a polar solvent, which upon injection will dramatically disturb the phase system characteristics of the SFC system. The phase-system switching approach, recently introduced for liquid chromatography-mass spectrometry, can be used to avoid this problem. Plasma samples containing the thermolabile and pH-sensitive cytostatic drug Mitomycin C (MMC) were injected onto a short precolumn. After washing and drying of the precolumn the compound of interest was desorbed using a supercritical fluid and analyzed by SFC. Up to 1 ml of plasma containing 20 ng of MMC has been analyzed in this way.

Title: Separation of opium alkaloids by

carbon dioxide sub- and supercritical fluid chromatography with packed columns. Application to the quantitative analysis of poppy straw extracts.

Abstract: The optimization of the separation of seven opium alkaloids by sub- and supercritical fluid chromatography (SFC) using packed columns and carbon dioxide as the primary mobile phase was studied. The influence of aminated polar modifiers on bare and aminopropyl-bonded silica was investigated. It was found that the presence of an amine in the mobile phase could enhance the retention of alkaloids on aminopropylsilica. Various separation schemes were possible depending

115

Authors: Janicot JL, Caude M, Rosset R


Address: Ecole Superieure de Physique et de Chimie de Paris, Laboratoire de Chimie Analytique France.

on the type of analysis needed. An aminopropyl-bonded silica used with a carbon dioxide-methanol-triethylamine-water mixture (82.95:16.25:0.50:0.30, w/w) gave a very rapid separation (2 min). A bare silica with a carbon dioxide-methanol-methylamine-water mixture (83.37:16.25:0.15:0.23, w/w) gave longer analysis (10 min) but a higher resolution. This last procedure was applied to a poppy straw extract and to the determination of three alkaloids of interest after a peak purity study using a diode-array UV detector. For these alkaloids, SFC appears to be a promising technique for the routine analysis of opium alkaloids.

Title: Comparison of packed column and

capillary column supercritical fluid chromatography and high-performance liquid chromatography using representative herbicides and pesticides as typical moderate polarity and molecular weight range molecules.

Authors: Wheeler JR, McNally ME


Address: Du Pont de Nemours & Co., Inc., Agricultural Products Department, Wilmington, DE 19898.

Abstract: The role of supercritical fluid chromatography (SFC) as a viable technique for analyzing agricultural products has been investigated using packed and capillary column methodology. The goal was to question the strengths and weaknesses of each of the techniques as possible approaches to separation strategies for thermally labile compounds as compared to the currently used high-performance liquid chromatography methodology. Representative herbicides have been examined with the techniques to compare such factors as linearity of response, limit of detection and reproducibility. In the literature so far, a direct numerical comparison of these three techniques for the analysis of real samples has not been presented. Results indicate that faster analyses, lower detection limits and greater injection to injection reproducibility are obtainable with packed column SFC. No appreciable difference in the linearity of response between the three techniques was noted. It has been well documented that the capillary SFC affords greater efficiencies and higher resolution [H. E. Schwartz, LC.GC, Mag. Liq. Gas Chromatogr., 5 (1987) 14]. Capillary SFC can also be interfaced to a variety of detectors not as easily accomplished with packed column SFC. Capillary SFC therefore still has a viable position in the analysis of low and moderate molecular weight and polarity molecules.

Title: Capillary supercritical fluid

chromatography of pyrrolizidine alkaloids.

Authors: Holzer G, Zalkow LH, Asibal CF

Abstract: The application of capillary supercritical fluid chromatography (SFC) for the separation of pyrrolizidine alkaloids (PAs) is examined. Most of these compounds are powerful toxins. Because they are present in at least 3% of all flowering plants, they are responsible for serious losses of


Address: Georgia Institute of Technology, School of Applied Biology, Atlanta 30332.

livestock and human disease. Capillary SFC, with its high separation efficiency, is shown to be an excellent method for the chromatographic analysis of complex PA mixtures. Complete separation of the PAs of the retronecine and otonecine family has been achieved by pressure-programmed capillary SFC. The operating conditions are mild enough to prevent thermal decomposition of the alkaloids during analysis.

Title: Resolution of enantiomeric

amides on a Pirkle-type chiral stationary phase. A comparison of subcritical fluid and liquid chromatographic approaches.

Authors: Macaudiere P, Tambute A, Caude M, Rosset R , Alembik MA, Wainer IW


Address: Ecole Superieure de Physique et de Chimie de Paris, Laboratoire de Chimie Analytique France.

Abstract: Subcritical and supercritical fluid chromatography (SubFC and SFC) have been evaluated for the resolution of an homologous series of enantiomeric amides. The solutes were the 2-naphthoyl amides of an homologous series of amines, ranging from 2-aminobutane to 2-aminoctane, and the p-methyl-, p-methoxy- and p-chlorophenylamides of 2-aminoheptane. The chiral stationary phase (CSP) used was the covalent form of (R)-N-(3,5-dinitrobenzoyl)phenylglycine. In liquid chromatography (LC) the mobile phase comprised hexane-2-propanol--acetonitrile (97:3:0.5) at a flow-rate of 2 ml/min and temperatures of 20-35 degrees C. In SFC, the mobile were various mixtures of carbon dioxide and polar modifiers, such as alcohols, chloroform and water. For the best conditions in LC, the chiral resolution, alpha, increased through the homologous series from alpha = 1.03 for the amide derived from 2-aminobutane to alpha = 1.11 for the 2-aminooctane amide. The values of alpha observed for the pi-basic amides of 2-aminoheptane (p-methyl and p-methoxy) were greater than that observed for the pi-acidic amide (p-chloro), i.e., alpha = 1.08 versus 1.04. The selectivities, resolutions and efficiencies obtained by LC and SubFC were similar. These results indicate that the mechanism of chiral recognition is the same in LC and SubFC and that the methods should be interchangeable. The actual analysis time for SubFC was significantly shorter than that required for LC: as short as 2 min for the 2-aminooctane amide, whereas LC takes over 10 min under the best conditions.

Title: Rapid and high resolution

capillary supercritical fluid chromatography (SFC) and SFC/MS of trichothecene

Abstract: The potential application of capillary column supercritical fluid chromatography (SFC) and SFC/mass spectrometry (SFC/MS) for the separation and analysis of mycotoxins of the trichothecene group was examined. Trichothecenes present significant analytical problems for both gas and liquid

117

mycotoxins. Authors: Smith RD, Udseth HR , Wright BW


Address:

chromatography with a major difficulty for the latter being the lack of sufficiently sensitive and selective detectors. Supercritical carbon dioxide mobile phases at temperatures up to 100 degrees C were used with deactivated fused silica columns coated with crosslinked stationary phases. Separations were obtained under pressure ramped conditions using long (15 m) 50-micron i.d. columns for several trichothecenes (diacetoxyscirpenol, deoxynivalenol, and T-2 toxin) and related higher molecular weight macrocyclic (roridin and verrucarin) trichothecenes. In addition, new rapid pressure programming techniques with short (less than 2m) 25- to 50-micron i.d. capillary columns were used to obtain fast separations in as little as 1 min. SFC/MS with ammonia chemical ionization provided high selectivity and sensitive detection (with approximately 1-pg detection limits) for trichothecene mixtures. The extension to complex sample matrices is discussed and the application of selective MS/MS detection is demonstrated.

Title: Soluble fibrin complexes and

fibrinogen heterogeneity in diabetes mellitus.

Authors: Tsianos EB, Stathakis NE

Journal: Thromb Haemost. 1980 44(3) 130-134

Address:

Abstract: The presence of soluble fibrin complexes (SFC) measured by gel filtration of plasma on 4% agarose columns, fibrinogen heterogeneity on 3.5% SDS-polyacrylamide gels and the concentrations of several plasma proteins were evaluated in 39 patients with diabetes mellitus (DM) and 19 matched control subjects. A small but significant increase of SFC was found in DM (p less than 0.01). On individual basis 51.2% of the patients had increased SFC (greater than M + 2 SD of the controls). Polyacrylamide gel electrophoresis of the SFC showed no evidence of cross-linking or proteolysis. Plasma clots formed in the presence of EDTA and trasylol were analysed in SDS-polyacrylamide gels in a normal and two lower molecular weight fibrin bands (band I, II, III). The percentage of band I fibrinogen was in diabetics (65.3 +/- 4.7%) lower than that of the controls (71.8 +/- 4.5%) (p less than 0.01). Fibrinogen levels, antithrombin III, alpha 1-antitrypsin, alpha 2-macroglobulin and plasminogen were significantly increased in DM. We suggest that in DM there is an enhancement of intravascular fibrin formation and accelerated fibrinogen degradation to lower molecular weight forms.

Title: A simple rapid method for

isolating soluble fibrin complexes Abstract: Using treatment with thrombin associated with trans-aminomethylcyclohexane carboxylic acid (t-AMCHA), a simple and rapid method for isolating soluble fibrin complexes (SFC) from fibrinogen in

from fibrinogen by treatment with thrombin and t-AMCHA.

Authors: Hayashi S, Yamada K

Journal: Thromb Haemost. 1980 42(5) 1388-1397

Address:

the plasma was developed. By this procedure, the recovery rates of SFC and early FDP (mainly X) increased according to the concentration of t-AMCHA, reaching a maximum at 286 mM t-AMCHA. On the other hand, the recovery rate of fibrinogen remained below 1.5% and that of late FDP was almost 100% at all concentrations of t-AMCHA. These results suggested that SFC and FDP could be isolated from fibrinogen by thrombin and t-AMCHA (286 MM) treatment. Moreover, it was possible to isolate SFC from FDP using gel filtration after treatment with thrombin and t-AMCHA. The SFC could be quantified by assay of the eluted fractions containing SFC by the staphylococcal clumping test.

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